Non-human carbonyl hydrolase mutants, DNA sequences and vectors encoding same and hosts transformed with said vectors

ABSTRACT

Novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and DNA sequences encoding the same. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in the amino acid sequence of a precursor carbonyl hydrolase. Such mutants have one or more properties which are different than the same property of the precursor hydrolase.

This is a continuation of application Ser. No. 08/212,291 filed Mar. 14, 1994 now U.S. Pat. No. 5,972,682, which is a continuation of application Ser. No. 07/898,382 filed Jun. 9, 1992, now abandoned which is a continuation of application Ser. No. 07/747,459 filed Aug. 12, 1991, now abandoned, which is a continuation of application Ser. No. 07/540,868 filed Jun. 14, 1990 now abandoned which is a continuation of application Ser. No. 07/035,652 filed Apr. 6, 1987, now abandoned which is a continuation-in-part of application Ser. No. 06/858,594 filed Apr. 30, 1986 now abandoned which is a continuation-in-part of applications Ser. Nos. 06/614,612, 06/614,615, 06/614,617 and 06/614,491, all filed May 29, 1984, each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and to DNA sequences encoding the same. Such mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in a precursor amino acid sequence.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolase. They comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. M. (1974) Sci Amer. 131, 74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct familites of enzymes: the Bacillus subtilisins and the mammalian and homologous bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.

Subtilisin is a serine endoprotease (MW 27,500) which is secreted in large amounts from a wide variety of Bacillus species. The protein sequence of subtilisin has been determined from at least four different species of Bacillus. Markland, F. S., et al. (1971) in The Enzymes, ed. Boyer P. D., Acad Press, New York, Vol. III, pp. 561-608; Nedkov, P. et al. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 1537-1540. The three-dimensional crystallographic structure of subtilisin BPN′ (from B. amyloligoefaciens) to 2.5A resolution has also been reported. Wright, C. S., et al. (1969) Nature 221, 235-242; Drenth, J. et al. (1972) Eur. J. Biochem. 26, 177-181. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972) Biochemistry 11, 2439-2449), product complexes (Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), and transition state analogs (Matthews, D. A., et al (1975) J. Biol. Chem. 250, 7120-7126; Poulos, T. L., et al. (1976) J. Biol. Chem. 251, 1097-1103), which have been reported have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin (Philipp, M., et al. (1983) Mol. Cell. Biochem. 51, 5-32; Svendsen, I. B. (1976) Carlsberg Res. Comm. 41, 237-291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methione at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965) J. Biol. Chem. 244, 5333-5338).

Substrate specificity is a ubiquitous feature of biological macromolecules that is determined by chemical forces including hydrogen bonding, electrostatic, hydrophobic and steric interactions. Jencks, W. P., in Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., in Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287. Substrate specificity studies of enzymes, however, have been limited to the traditional means of probing the relative importance of these binding forces. Although substrate analogs can be synthesized chemically, the production of modified enzyme analogs has been limited to chemically modified enzyme derivatives (Kaiser, E. T., et al. (1985) Ann. Rev. Biochem. 54, 565-595 or naturally occurring mutants. Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358.

The recent development of various in vitro techniques to manipulate the DNA sequences encoding naturally-occuring polypeptides as well as recent developments in the chemical synthesis of relatively short sequences of single and double stranded DNA has resulted in the speculation that such techniques can be used to modify enzymes to improve some functional property in a predictable way. Ulmer, K. M. (1983) Science 219, 666-671. The only working example disclosed therein, however, is the substitution of a single amino acid within the active site of tyrosyl-tRNA synthetase (Cys35→Ser) which lead to a reduction in enzymatic activity. See Winter, G., et al. (1982) Nature 299, 756-758; and Wilkinson, A. J., et al. (1983) Biochemistry 22, 3581-3586 (Cys35→Gly mutation also resulted in decreased activity).

When the same t-RNA synthetase was modified by substituting a different amino acid residue within the active site with two different amino acids, one of the mutants (Thr51→Ala) reportedly demonstrated a predicted moderate increase in kcat/Km whereas a second mutant (Thr51→Pro) demonstrated a massive increase in kcat/Km which could not be explained with certainty. Wilkinson, A. H., et al. (1984) Nature 307, 187-188.

Another reported example of a single substitution of an amino acid residue is the substitution of cysteine for isoleucine at the third residue of T4 lysozyme. Perry, L. J., et al. (1984) Science 226, 555-557. The resultant mutant lysozyme was mildly oxidized to form a disulfide bond between the new cysteine residue at position 3 and the native cysteine at position 97. This crosslinked mutant was initially described by the author as being enzymatically identical to, but more thermally stable than, the wild type enzyme. However, in a “Note Added in Proof”, the author indicated that the enhanced stability observed was probably due to a chemical modification of cysteine at residue 54 since the mutant lysozyme with a free thiol at Cys54 has a thermal stability identical to the wild type lysozyme.

Similarly, a modified dehydrofolate reductase from E. coli has been reported to be modified by similar methods to introduce a cysteine which could be crosslinked with a naturally-occurring cysteine in the reductase. Villafranca, D. E., et al. (1983) Science 222, 782-788. The author indicates that this mutant is fully reactive in the reduced state but has significantly diminished activity in the oxidized state. In addition, two other substitutions of specific amino acid residues are reported which resulted in mutants which had diminished or no activity.

As set forth below, several laboratories have also reported the use of site directed mutagensis to produce the mutation of more than one amino acid residue within a polypeptide.

The amino-terminal region of the signal peptide of the prolipoprotein of the E. coli outer membrane was stated to be altered by the substitution or deletion of residues 2 and 3 to produce a charge change in that region of the polypeptide. Inoyye, S., et al. (1982) Proc. Nat. Acad. Sci. USA 79, 3438-3441. The same laboratory also reported the substitution and deletion of amino acid redisues 9 and 14 to determine the effects of such substitution on the hydrophobic region of the same signal sequence. Inouye, S., et al. (1984) J. Biol. Chem. 259, 3729-3733. In the case of mutants at residues 2 and 3 the authors state that the results obtained were consistant with the proposed loop model for explaining the functions of the signal sequence. However, as reported the mutations at residues 9 and 14 produced results indicating that the signal peptide has unexpeded flexibility in terms of the relationship between its primary structure and function in protein secretion.

Double mutants in the active site of tyrosyl-t-RNA synthetase have also been reported. Carter, P. J., et al. (1984) Cell 38, 835-840. In this report, the improved affinity of the previously described Thr51→Pro mutant for ATP was probed by producing a second mutation in the active site of the enzyme. One of the double mutants, Gly35/Pro51, reportedly demonstrated an unexpected result in that it bound ATP in the transition state better than was expected from the two single mutants. Moreover, the author warns, at least for one double mutant, that it is not readily predictable how one substitution alters the effect caused by the other substitution and that care must be taken in interpreting such substitutions.

A mutant is disclosed in U.S. Pat. No. 4,532,207, wherein a polyarginine tail was attached to the C-terminal residue of β-urogastrone by modifying the DNA sequence encoding the polypeptide. As disclosed, the polyarginine tail changed the electrophoretic mobility of the urogastrone-polyaginine hybrid permiting selective purification. The polyarginine was subsequently removed, according to the patentee, by a polyarginine specific exopeptidase to produce the purified urogastrone. Properly construed, this reference discloses hybrid polypeptides which do not constitute mutant polypeptides containing the substitution, insertion or deletion of one or more amino acids of a naturally occurring polypeptide.

Single and double mutants of rat pancreatic trypsin have also been reported. Craik, C. S., et al. (1985) Science 228, 291-297. As reported, glycine residues at positions 216 and 226 were replaced with alanine residues to produce three trypsin mutants (two single mutants and one double mutant). In the case of the single mutants, the authors stated expectation was to observe a differential effect on Km. They instead reported a change in specificity (kcat/Km) which was primarily the result of a decrease in kcat. In contrast, the double mutant reportedly demonstrated a differential increase in Km for lysyl and arginyl substrates as compared to wild type trypsin but had virtually no catalytic activity.

The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications.

Based on the above references, however, it is apparent that the modification of the amino acid sequence of wild type enzymes often results in the decrease or destruction of biological activity. Moreover, these references do not address the mutation of the particular carbonyl hydrolases disclosed herein.

Accordingly, it is an object herein to provide carbonyl hydrolase mutants which have at least one property which is different from the same property of the carbonyl hydrolase precursor from which the amino acid of said mutant is derived.

It is a further object to provide mutant DNA sequences encoding such carbonyl hydrolase mutants as well as expression vectors containing such mutant DNA sequences.

Still further, another object of the present invention is to provide host cells transformed with such vectors as well as host cells which are capable of expressing such mutants either intracellularly or extracellularly.

SUMMARY OF THE INVENTION

The invention includes carbonyl hydrolase mutants, preferably having at least one property which is substantially different from the same property of the precursor non-human carbonyl hydrolase from which the amino acid sequence of the mutant is derived. These properties include oxidative stability, substrate, specificity catalytic activity, thermal stability, alkaline stability, pH activity profile and resistance to proteolytic degradation. The precursor carbonyl hydrolase may be naturally occurring carbonyl hydrolases or recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is derived by the substitution, deletion or insertion of one or more amino acids of the precursor carbonyl hydrolase amino acid sequence.

The invention also includes mutant DNA sequences encoding such carbonyl hydrolase mutants. These mutant DNA sequences are derived from a precursor DNA sequence which encodes a naturally occurring or recombinant precursor carbonyl hydrolase. The mutant DNA sequence is derived by modifying the precursor DNA sequence to encode the substitution, deletion or insertion of one or more amino acids encoded by the precursor DNA sequence. These recombinant DNA sequences encode mutants having an amino acid sequence which does not exist in nature and at least one property which is substantially different from the same property of the precursor carbonyl hydrolase encoded by the precursor DNA sequence.

Further the invention includes expression vectors containing such mutant DNA sequences as well as host cells transformed with such vectors which are capable of expressing said carbonyl hydrolase mutants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the nucleotide sequence of the coding strand, correlated with the amino acid sequence of B. amyloliguefaciens subtilisin gene. Promoter (p) ribosome binding site (rbs) and termination (term) regions of the DNA sequence as well as sequences encoding the presequence (PRE) putative prosequence (PRO) and mature form (MAT) of the hydrolase are also shown.

FIG. 2 is a schematic diagram showing the substrate binding cleft of subtilisin together with substrate.

FIG. 3 is a stereo view of the S-1 binding subsite of B. amyloliguefaciens subtilisin showing a lysine P-1 substrate bound in the site in two different ways. FIG. 3A shows Lysine P-1 substrate bound to form a salt bridge with a Glu at position 156. FIG. 3B shows Lysine P-1 substrate bound to form a salt bridge with Glu at position 166.

FIG. 4 is a schematic diagram of the active site of subtilisin Asp32, His64 and Ser221.

FIGS. 5A-1, 5A-2, 5B-1, and 5B-2 depict the amino acid sequence of subtilisin obtained from various sources. The residues directly beneath each residue of B. amyloliguefaciens subtilisin are equivalent residues which (1) can be mutated in a similar manner to that described for B. amyloliguefaciens subtilisin, or (2) can be used as a replacement amino acid residue in B. amyloliguefaciens subtilisin. FIG. 5C depicts conserved residues of B. amyloliguefaciens subtilisin when compared to other subtilisin sequences.

FIGS. 6A and 6B depict the inactivation of the mutants Met222L and Met222Q when exposed to various organic oxidants.

FIGS. 7A and 7B depict the ultraviolet spectrum of Met222F subtilisin and the difference spectrum generated after inactivation by diperdodecanoic acid (DPDA).

FIG. 8 shows the pattern of cyanogen bromide digests of untreated and DPDA oxidized subtilisin Met222F on high resolution SDS-pyridine peptide gels.

FIG. 9 depicts a map of the cyanogen bromide fragments of FIG. 8 and their alignment with the sequence of subtilisin Met222F.

FIG. 10 depicts the construction of mutations between codons 45 and 50 of B. amyloliguefaciens subtilisin.

FIG. 11 depicts the construction of mutations between codons 122 and 127 of B. amyloliguefaciens subtilisin.

FIG. 12 depicts the effect of DPDA on the activity of subtilisin mutants at positions 50 and 124 in subtilisin Met222F.

FIG. 13 depicts the construction of mutations at codon 166 of B. amyloliguefaciens subtilisin.

FIG. 14 depicts the effect of hydrophobicity of the P-1 substrate side-chain on the kinetic parameters of wild-type B. amyloliguefaciens subtilisin.

FIG. 15 depicts the effect of position 166 side-chain substitutions on P-1 substrate specificity. FIG. 15A shows position 166 mutant subtilisins containing non-branched alkyl and aromatic side-chain substitutions arranged in order of increasing molecular volume. FIG. 15B shows a series of mutant enzymes progressing through β- and γ-branched aliphatic side chain substitutions of increasing molecular volume.

FIGS. 16A, 16B, 16C and 16D depict the effect of position 166 side-chain volumn on log kcat/Km for various P-1 substrates.

FIG. 17 shows the substrate specificity differences between Ile166 and wild-type (Gly166) B. amyloliguefaciens subtilisin against a series of alphatic and aromatic substrates. Each bar represents the difference in log kcat/Km for Ile166 minus wild-type (Gly166) subtilisin.

FIG. 18 depicts the construction of mutations at codon 169 of B. amyloliguefaciens subtilisin.

FIG. 19 depicts the construction of mutations at codon 104 of B. amyloliguefaciens subtilisin.

FIG. 20 depicts the construction of mutations at codon 152 B. amyloliguefaciens subtilisin.

FIG. 21 depicts the construction of single mutations at codon 156 and double mutations at codons 156 and 166 of B. amyloliguefaciens subtilisin.

FIG. 22 depicts the construction of mutations at codon 217 for B. amyloliguefaciens subtilisin:

FIG. 23A depicts the kcat/Km versus pH profile for mutations at codon 156 and 166 in B. amyloliguefaciens subtilisin.

FIG. 23B depicts the kcat/Km versus pH profile for mutations at codon 156 and 166 in B. amyloliguefaciens subtilisin.

FIG. 24 depicts the kcat/Km versus pH profile for mutations at codon 222 in B. amyloliguefaciens subtilisin.

FIG. 25 depicts the constructing mutants at codons 94, 95 and 96.

FIGS. 26 and 27 depict substrate specificity of proteins for 4 substrates.

FIGS. 28 A, B, C and D depict the effect of charge in the P-1 binding sites due to substitutions at codon 156 and 166.

FIGS. 29 A and B are a stereoview of the P-1 binding site of subtilisin BPN′ showing a lysine P-1 substrate bound in the site in two ways. In 29A, Lysine P-1 substrate is built to form a salt bridge with a Glu at codon 156. In 29B, Lysine P-1 substrate is built to form a salt bridge with Glu at codon 166.

FIGS. 30A, 30B, and 30C demonstrate residual enzyme activity versus temperature curves for purified wild-type (Panel A), C22/C87 (Panel B) and C24/C87 (Panel C).

FIG. 31 depicts the strategy for producing point mutations in the subtilisin coding sequence by misincorporation of α-thioldeoxynucleotide triphosphates.

FIG. 32 depicts the autolytic stability of purified wild type and mutant subtilisins 170E, 107V, 213R and 107V/213R at alkaline pH.

FIG. 33 depicts the autolytic stability of purified wild type and mutant subtilisins V50, F50 and F50/V107/R213 at alkaline pH.

FIG. 34 depicts the strategy for constructing plasmids containing random cassette mutagenesis over residues 197 through 228.

FIG. 35A and 35B depict the oligodeoxynucleotides used for random cassette mutagenesis over residues 197 through 228.

FIG. 36 depicts the construction of mutants at codon 204.

FIG. 37 depicts the oligodeoxynucleotides used for synthesizing mutants at codon 204.

DETAILED DESCRIPTION

The inventors have discovered that various single and multiple in vitro mutations involving the substitution, deletion or insertion of one or more amino acids within a non-human carbonyl hydrolase amino acid sequence can confer advantageous properties to such mutants when compared to the non-mutated carbonyl hydrolase.

Specifically, B. amyloliguefaciens subtilisin, an alkaline bacterial protease, has been mutated by modifying the DNA encoding the subtilisin to encode the substitution of one or more amino acids at various amino acid residues within the mature form of the subtilisin molecule. These in vitro mutant subtilisins have at least one property which is different when compared to the same property of the precursor subtilisin. These modified properties fall into several categories including: oxidative stability, substrate specificity, thermal stability, alkaline stability, catalytic activity, pH activity profile, resistance to proteolytic degradation, Km, kcat and Km/kcat ratio.

Carbonyl hydrolases are enzymes which hydrolyze compounds containing

bonds in which X is oxygen or nitrogen. They include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. Naturally occurring carbonyl hydrolases principally include hydrolases, e.g. lipases and peptide hydrolases, e.g. subtilisins or metalloproteases. Peptide hydrolases include α-aminoacylpeptide hydrolase, peptidylamino-acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid proteases are included, as well as endo and exoproteases.

“Recombinant carbonyl hydrolase” refers to a carbonyl hydrolase in which the DNA sequence encoding the naturally occurring carbonyl hydrolase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the carbonyl hydrolase amino acid sequence. Suitable modification methods are disclosed herein and in EPO Publication No. 0130756 published Jan. 9, 1985.

Subtilisins are bacterial carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, “subtilisin” means a naturally occurring subtilisin or a recombinant subtilisin. A series of naturally occurring subtilisins is known to be produced and often secreted by various bacterial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus is aspartate-histidine-serine. In the chymotrypsin related proteases tne relative order, however is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.

“Recombinant subtilisin” refers to a subtilisin in which the DNA sequence encoding the subtilisin is modified to produce a mutant DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally occurring subtilisin amino acid sequence. Suitable methods to produce such modification include those disclosed herein and in EPO Publication No. 0130756. For example, the subtilisin multiple mutant herein containing the substitution of methionine at amino acid residues 50, 124 and 222 with phenylalanine, isoleucine and glutamine, respectively, can be considered to be derived from the recombinant subtilisin containing the substitution of glutamine at residue 222 (Gln222) disclosed in EPO Publication No. 0130756. The multiple mutant thus is produced by the substitution of phenylalanine for methionine at residue 50 and isoleucine for methionine at residue 124 in the Gln222 recombinant subtilisin.

“Non-human carbonyl hydrolases” and their genes may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as E. coli or pseudomonas and gram positive bacteria such as micrococcus or bacillus. Examples of eucaryotic organisms from which carbonyl hydrolase and their genes may be obtained include yeast such as S. cerevisiae, fungi such as Aspergillus sp., and non-human mammalian sources such as, for example, Bovine sp. from which the gene encoding the carbonyl hydrolase chymosin can be obtained. As with subtilisins, a series of carbonyl hydrolases can be obtained from various related species which have amino acid sequences which are not entirely homologous between the members of that series but which nevertheless exhibit the same or similar type of biological activity. Thus, non-human carbonyl hydrolase as used herein has a functional definition which refers to carbonyl hydrolases which are associated, directly or indirectly, with procaryotic and non-human eucaryotic sources.

A “carbonyl hydrolase mutant” has an amino acid sequence which is derived from the amino acid sequence of a non-human “precursor carbonyl hydrolase”. The precursor carbonyl hydrolases include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is “derived” from the precursor hydrolase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the “precursor DNA sequence” which encodes the amino acid sequence of the precursor carbonyl hydrolase rathern than manipulation of the precursor carbonyl hydrolase per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in EPO Publication No. 0130756.

Specific residues of B. amyloliguefaciens subtilisin are identified for substitution, insertion or deletion. These amino acid position numbers refer to those assigned to the B. amyloliguefaciens subtilisin sequence presented in FIG. 1. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor carbonyl hydrolases containing amino acid residues which are “equivalent” to the particular identified residues in B. amyloliguefaciens subtilisin.

A residue (amino acid) of a precursor carbonyl hydrolase is equivalent to a residue of B. amyloliguefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analagous to a specific residue or portion of that residue in B. amyloliguefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor carbonyl hydrolase is directly comparted to the B. amyloliguefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in all subtilisins for which sequence is known (FIG. 5C). After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of B. amyloliguefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained.

For example, in FIG. 5A the amino acid sequence of subtilisin from B. amyloliguefaciens B. subtilisin var. I168 and B. lichenformis (carlsbergensis) are aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. These residues are identified in FIG. 5C.

These conserved residues thus may be used to define the corresponding equivalent amino acid residues of B. amyloliguefaciens subtilisin in other carbonyl hydrolases such as thermitase derived from

Thermoactinomyces. These two particular sequences are aligned in FIG. 5B to produce the maximum homology of conserved residues. As can be seen there are a number of insertions and deletions in the thermitase sequence as compared to B. amyloliguefaciens subtilisin. Thus, the equivalent amino acid of Tyr217 in B. amyloliguefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217.

In FIG. 5A, the equivalent amino acid at position 217 in B. amyloliguefaciens subtilisin is Tyr. Likewise, in B. subtilis subtilisin position 217 is also occupied by Tyr but in B. licheniformis position 217 is occupied by Leu.

Thus, these particular residues in thermitase, and subtilisin from B. subtilisin and B. licheniformis may be substituted by a different amino acid to produce a mutant carbonyl hydrolase since they are equivalent in primary structure to Tyr217 in B. amyloliguefaciens subtilisin. Equivalent amino acids of course are not limited to those for Tyr217 but extend to any residue which is equivalent to a residue in B. amyloliguefaciens whether such residues are conserved or not.

Equivalent residues homologous at the level of tertiary structure for a precursor carbonyl hydrolase whose tertiary structure has been determined by x-ray crystallography, are defined as those for which the atomic coordinates of 2 or more of the main chain atoms of a particular amino acid residue of the precursor carbonyl hydrolase and B. amyloliguefaciens subtilisin (N on N, CA on CA, C on C, and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the carbonyl hydrolase in question to the B. amyloliguefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. ${R\quad {factor}} = \frac{\sum\limits_{h}\left| {{Fo}(h)} \middle| {- \left| {{Fc}(h)} \right|} \right.}{\sum\limits_{h}\left| {{Fo}(h)} \right|}$

Equivalent residues which are functionally analogous to a specific residue of B. amyloliguefaciens subtilisin are defined as those amino acids of the precursor carbonyl hydrolases which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the B. amyloliguefaciens subtilisin as described herein. Further, they are those residues of the precursor carbonyl hydrolase (for which a tertiary structure has been obtained by x-ray crystallography), which occupy an analogous position to the extent that although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of B. amyloliguefaciens subtilisin. The three dimensional structures would be aligned as outlined above.

Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a mutant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in a naturally occurring sequence. The carbonyl hydrolase mutants of the present invention include the mature forms of carbonyl hydrolase mutants as well as the pro- and prepro-forms of such hydrolase mutants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the carbonyl hydrolase mutants.

“Prosequence” refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a carbonyl hydrolase which when removed results in the appearance of the “mature” form of the carbonyl hydrolase. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. The preferred prosequence for producing carbonyl hydrolase mutants, specifically subtilisin mutants, is the putative prosequence of B. amyloliguifaciens subtilisin although other subtilisin prosequences may be used.

A “signal sequence” or “presequence” refers to any sequence of amino acids bound to the N-terminal portion of a carbonyl hydrolase or to the N-terminal portion of a prohydrolase which may participate in the secretion of the mature or pro forms of the hydrolase. This definition of signal sequence is a functional one, meant to include all those amino acid sequences, encoded by the N-terminal portion of the subtilisin gene or other secretable carbonyl hydrolases, which participate in the effectuation of the secretion of subtilisin or other carbonyl hydrolases under native conditions. The present invention utilizes such sequences to effect the secretion of the carbonyl hydrolase mutants as defined herein.

A “prepro” form of a carbonyl hydrolase mutant consists of the mature form of the hydrolase having a prosequence operably linked to the amino-terminus of the hydrolase and a “pre” or “signal” sequence operably linked to the amino terminus of the prosequence.

“Expression vector” refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

The “host cells” used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in EPO Publication No. 0130756 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing subtilisin is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in EPO Publicatin No. 0130756 and further described by Yang, M. Y., et al. (1984) J. Bacteriol. 160, 15-21. Such host cells are distinguishible from those disclosed in PCT Publication No. 03949 wherein enzymatically inactive mutants of intracellular proteases in E. coli are disclosed. Other host cells for expressing subtilisin include Bacillus subtilis I168 (EPO Publication No. 0130756).

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the carbonyl hydrolase mutants or expressing the desired carbonyl hydrolase mutant. In the case of vectors which encode the pre or prepro form of the carbonyl hydrolase mutant, such mutants, when expressed, are typically secreted from the host cell into the host cell medium.

“Operably linked” when describing the relationship between two DNA regions simply means that they are functionally related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

The genes encoding the naturally-occurring precursor carbonyl hydrolase may be obtained in accord with the general methods described in EPO Publication No. 0130756. As can be seen from the examples disclosed therein, the methods generally comprise synthesizing labelled probes having putative sequences encoding regions of the hydrolase of interest, preparing genomic libraries from organisims expressing the hydrolase, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The cloned carbonyl hydrolase is then used to transform a host cell in order to express the hydrolase. The hydrolase gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promoter if it is recognized, i.e., transcribed, by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the hydrolase gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the hydrolase gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosonal limitations. However, it is within the scope herein to integrate multiple copies of the hydrolase gene into host genome. This is facilitated by procaryotic and eucaryotic organisms which are particularly susceptible to homologous recombination.

Once the carbonyl hydrolase gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor carbonyl hydrolase. Such modifications include the production of recombinant carbonyl hydrolases as disclosed in EPO Publication No. 0130756 and the production of carbonyl hydrolase mutants described herein.

The following cassette mutagenesis method may be used to facilitate the construction and identification of the carbonyl hydrolase mutants of the present invention although other methods including site-directed mutagenesis may be used. First, the gene encoding the hydrolase is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the expressed enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the hydrolase gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the hydrolase gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.

Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. Once the gene is cloned, the restriction sites flanking the sequence to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is enormously simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

The number of commercially available restriction enzymes having sites not present in the gene of interest is generally large. A suitable DNA sequence computer search program simplifies the task of finding potential 5′ and 3′ convenient flanking sites. A primary constraint is that any mutation introduced in creation of the restriction site must be silent to the final construction amino acid coding sequence. For a candidate restriction site 5′ to the target codon a sequence must exist in the gene which contains at least all the nucleotides but for one in the recognition sequence 5′ to the cut of the candidate enzyme. For example, the blunt cutting enzyme SmaI (CCC/GGG) would be a 5′ candidate if a nearby 5′ sequence contained NCC, CNC, or CCN. Furthermore, if N needed to be altered to C this alteraiton must leave the amino acid coding sequence intact. In cases where a permanent silent mutation is necessary to introduce a restriction site one may want to avoid the introduction of a rarely used codon. A similar situation of SmaI would apply for 3′ flanking sites except the sequence NGG, GNG, or GGN must exist. The criteria for locating candidate enzymes is most relaxed for blunt cutting enzymes and most stringent for 4 base overhang enzymes. In general many candidate sites are available. For the codon-221 target described herein a BalI site (TGG/CCA) would have been engineered in one base pair 5′ from the KpnI site. A 3′ EcoRV site (GAT/ATC) could have been employed 11 base pairs 5′ to the PstI site. A cassette having termini ranging from a blunt end up to a four base-overhang will function without difficulty. In retrospect, this hypothetical EcoRV site would have significantly shortened the oligonucleotide cassette employed (9 and 13 base pairs) thus allowing greater purity and lower pool bias problems. Flanking sites should obviously be chosen which cannot themselves ligate so that ligation of the oligonucleotide cassette can be assured in a single orientation.

The mutant carbonyl hydrolases expressed upon transformation of suitable hosts are screened for enzymes exhibiting one or more properties which are substantially different from the properties of the precursor carbonyl hydrolases, e.g., changes in substrate specificity, oxidative stability, thermal stability, alkaline stability, resistance to proteolytic degradation, pH-activity profiles and the like.

The carbonyl hydrolase mutants of the present invention may also be generated by random mutagenesis. See for example the methods disclosed by Shortle, D., et al. (1985) Genetics, 110, 539; Shortle, D., et al. (1986) Proteins: Structure, Function and Genetics, 1, 81; Shortle, D. (1986) J. Cell. Biochem, 30, 281; Alber, T., et al. (1985) Proc. Natl. Acad. of Sci., 82, 747; Matsumura, M., et al. (1985) J. Biochem., 260, 15298; Liao, H., et al. (1986) Proc. Natl. Acad. of Sci., 83 576; and the random mutagenesis method disclosed herein.

When combined with the alkaline stability screening procedure disclosed herein, mutants obtained by random mutagenesis were identified which demonstrated either increased or decreased alkaline or thermal stability.

A change in substrate specificity is defined as a difference between the kcat/Km ratio of the precursor carbonyl hydrolase and that of the hydrolase mutant. The kcat/Km ratio is a measure of catalytic efficienty. Carbonyl hydrolase mutants with increased or diminished kcat/Km ratios are described in the examples. Generally, the objective will be to secure a mutant having a greater (numerically large) kcat/Km ratio for a given substrate, thereby enabling the use of the enzyme to more efficiently act on a target substrate. A substantial change in kcat/Km ratio is preferably at least 2-fold increase or decrease. However, smaller increases or decreases in the ratio (e.g., at least 1.5-fold) are also considered substantial. An increase in kcat/Km ratio for one substrate may be accompanied by a reduction in kcat/Km ratio for another substrate. This is a shift in substrate specificity, and mutants exhibiting such shifts have utility where the precursor hydrolase is undesirable, e.g. to prevent undesired hydrolysis of a particular substrate in an admixture of substrates. Km and kcat are measured in accord with known procedures, as described in EPO Publication No. 0130756 or as described herein.

Oxidative stability is measured either by known procedures or by the methods described hereinafter. A substantial change in oxidative stability is evidenced by at least about 50% increase or decrease (preferably decrease) in the rate of loss of enzyme activity when exposed to various oxidizing conditions. Such oxidizing conditions are exposure to the organic oxidant diperdodecanoic acid (DPDA) under the conditions described in the examples.

Alkaline stability is measured either by known procedures or by the methods described herein. A substantial change in alkaline stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half life of the enzymatic activity of a mutant when compared to the precursor carbonyl hydrolase. In the case of subtilisins, alkaline stability was measured as a function of autoproteolytic degradation of subtilisin at alkaline pH, e.g. for example, 0.1M sodium phosphate, pH 12 at 25° or 30° C.

Thermal stability is measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half-life of the catalytic activity of a mutant when exposed to a relatively high temperature and neutral pH as compared to the precursor carbonyl hydrolase. In the case of subtilisins, thermal stability is measured by the autoproteolytic degradation of subtilisin at elevated temperatures and neutral pH, e.g., for example 2 mM calcium chloride, 50 mM MOPS pH 7.0 at 59° C.

The inventors have produced mutant subtilisins containing the substitution of the amino acid residues of B. amyloliguefaciens subtilisin shown in Table I. The wild type amino acid sequence and DNA sequence of B. amyloliguefaciens subtilisin is shown in FIG. 1.

TABLE I Residue Replacement Amino Acid Tyr2l F Thr22 C Ser24 C Asp32 N Q S Ser33 A T Asp36 A G Gly46 V Ala48 E V R Ser49 C L Met50 C F V Asn77 D Ser87 C Lys94 C Val95 C Tyrl04 A C D E F G H I K L M N P Q R S T V W Ile107 V Gly110 C R Met124 I L Ala152 G S Asn155 A D H Q T Glu156 Q S Gly166 A C D E F H I K L M N P Q R S T V W Y Gly169 A C D E F H I K L M N P Q R S T V W Y Lys170 E R Tyr17l F Pro172 E Q Phe189 A C D E G H I K L M N P Q R S T V W Y Asp197 R A Met199 I Ser204 C R L P Lys213 R T Tyr2l7 A C D E F G H I K L M N P Q R S T V W Ser221 A C Met222 A C D E F G H I K L N P Q R S T V W Y

The different amino acids substituted are represented in Table I by the following single letter designations:

Amino acid or residue 3-letter 1-letter thereof symbol symbol Alanine Ala A Glutamate Glu E Glutamine Gln Q Aspartate Asp D Asparagine Asn N Leucine Leu L Glycine Gly G Lysine Lys K Serine Ser S Valine Val V Arginine Arg R Threonine Thr T Proline Pro P Isoleucine Ile I Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y Cysteine Cys C Tryptophan Trp W Histidine His H

Except where otherwise indicated by context, wild-type amino acids are represented by the above three-letter symbols and replaced amino acids by the above single-letter symbols. Thus, if the methionine at residue 50 in B. amyloliguefaciens subtilisin is replaced by Phenylalanine, this mutation (mutant) may be designated Met50F or F50. Similar designations will be used for multiple mutants.

In addition to the amino acids used to replace the residues disclosed in Table I, other replacements of amino acids at the residues are expected to produce mutant subtilisins having useful properties. These residues and replacement amino acids are shown in Table II.

TABLE II Residue Replacement Amino Acid(s) Tyr-21 L Thr22 K Ser24 A Asp32 Ser33 G Gly46 Ala48 Ser49 Met50 L K I V Asn77 D Ser87 N Lys94 R Q Val95 L I Tyr104 Met124 K A Ala152 C L I T M Asn155 Glu156 A T M L Y Gly166 Gly169 Tyr171 K R E Q Pro172 D N Phe189 Tyr217 Ser221 Met222

Each of the mutant subtilisins in Table I contain the replacement of a single residue of the B. amyloliguefaciens amino acid sequence. These particular residues were chosen to probe the influence of such substitutions on various properties of B. amyloliguefacien subtilisin.

Thus, the inventors have identified Met124 and Met222 as important residues which if substituted with another amino acid produce a mutant subtilisin with enhanced oxidative stability. For Met124, Leu and Ile are preferred replacement amino acids. Preferred amino acids for replacement of Met222 are disclosed in EPO Publication No. 0130756.

Various other specific residues have also been identified as being important with regard to substrate specificity. These residues include Tyr104, Ala152, Glu156, Gly166, Gly169, Phe189 and Tyr217 for which mutants containing the various replacement amino acids presented in Table I have already been made, as well as other residues presented below for which mutants have yet to be made.

The identification of these residues, including those yet to be mutated, is based on the inventors high resolution crystal structure of B. amyloliguefaciens subtilisin to 1.8 A (see Table III), their experience with in vitro mutagenesis of subtilisin and the literature on subtilisin. This work and the above referenced x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors, product complexes and transition state analogs has helped in identifying an extended peptide binding cleft in subtilisin. This substrate binding cleft together with substrate is schematically diagramemed in FIG. 2, according to the nomenclature of Schechter, I., et al. (1967) Biochem Bio. Res. Commun. 27, 157. The scissile bond in the substrate is identified by an arrow. The P and P′ designations refer to the amino acids which are positioned respectively toward the amino or carboxy terminus relative to the scissle bond. The S and S′ designations refer to subsites in the substrate binding cleft of subtilisin which interact with the corresponding substrate amino acid residues.

Atomic Coordinates for the Apoenzyme Form of B, Amyloliguefaciens Subtilisin to 1.8A Resolution 1 ALA N 19.434 53.195 -21.756 1 ALA CA 19.811 51.774 -21.965 1 ALA C 18.731 58.995 -21.324 1 ALA D 18.376 51.197 -20.175 1 ALA CB 21.099 52.518 -21.183 2 GLN N 18.268 49.886 -22.841 2 GLN CA 17.219 49.008 -21.434 2 GLN C 17.875 47.706 -28.992 2 GLN D 18.765 47.165 -21.691 2 GLN CB 16.125 48.768 -22.449 2 GLN CG 15.028 47.905 -21.927 2 GLN CD 13.912 47.762 -22.930 2 GLN DE1 13.O23 48.612 -22.867 2 GLN UEZ 14.115 46.917 -23.926 3 SER N 17.477 47.205 -19.852 3 SER CA 17.958 45.868 -19.437 3 SER C 16.735 44.918 -19.498 3 SER D 15.598 45.352 -19.229 3 SER CB 18.588 45.838 -18.069 3 SER DG 17.682 46.218 -17.849 4 VAL N 16.991 43.646 -19.725 4 VAL CA 15.946 42.619 -19.639 4 VAL C 16.129 41.934 -18.298 4 VAL D 17.123 41.178 -18.886 4 VAL CB 16.008 41.622 -20.822 4 VAL CG1 14.874 48.572 -28.741 4 VAL CG2 16.037 42.266 -22.186 5 PRO N 15.239 42.106 -17.331 5 PRO CA 15.384 41.415 -16.827 5 PRO C 15.581 39.905 -16.249 5 PRO O 14.885 39.263 -17.146 5 PRO CB 14.150 41.880 -15.243 5 PRO CG 13.841 43.215 -15.921 5 PRO CD 14.044 42.986 -17.417 6 TYR N 16.343 39.240 -15.487 6 TYR CA 16.628 37.803 -15.715 6 TYR C 15.359 36.975 -15.528 6 TYR D 15.224 35.943 -16.235 6 TYR CB 17.824 37.323 -14.834 6 TYR CG 18.021 35.847 -15.055 6 TYR CD1 18.437 35.452 -16.346 6 TYR CD2 17.696 34.908 -14.871 6 TYR CE1 18.535 34.870 -16.653 6 TYR CE2 17.815 33.539 -14.379 6 TYR CZ 18.222 33.154 -15.628 6 TYR OH 18.312 31.838 -15.996 7 GLY N 14.464 37.362 -14.638 7 GLY CA 13.211 36.640 -14.376 7 GLY C 12.400 36.535 -15.678 7 GLY O 11.747 35.478 -15.883 8 VAL N 12.441 37.529 -16.541 8 VAL CA 11.777 37.523 -17.836 8 VAL C 12.363 36.433 -18.735 8 VAL O 11.639 35.716 -19.47O 8 VAL CB 11.765 38.900 -18.567 8 VAL CG1 11.106 38.893 -19.943 8 VAL CG2 18.991 39.919 -17.733 9 SER N 13.661 36.318 -18.775 9 SER CA 14.419 35.342 -19.562 9 SER C 14.188 33.920 -18.965 9 SER O 14.112 33.014 -19.801 9 SER CB 15.926 35.632 -19.505 9 SER OG 14.162 36.747 -20.358 10 GLN N 14.115 33.887 -17.662 10 GLN CA 13.964 32.636 -16.876 10 GLN C 12.687 31.887 -17.277 10 GLN O 12.785 3O.642 -17.413 10 GLN CB 14.125 32.885 -15.418 10 GLN CG 14.295 31.617 -14.588 10 GLN CD 14.486 31.911 -13.147 10 GLN OE1 14.554 33.868 -12.744 10 GLN NE2 14.552 30.960 -12.251 11 ILE N 11.625 32.575 -17.678 11 ILE CA 10.373 31.904 -18.182 11 ILE C 10.209 31.792 -19.605 11 ILE O 9.173 31.333 -20.180 11 ILE CB 9.132 32.669 -17.475 11 ILE CG1 9.066 34.117 -18.849 11 ILE CG2 9.162 32.655 -15.941 11 ILE CO1 7.588 34.648 -17.923 12 LYS N 11.272 32.185 -20.277 12 LYS CA 11.388 32.119 -21.722 12 LYS C 10.454 33.806 -22.522 12 LYS O 10.173 32.703 -23.686 12 LYS CB 11.257 30.646 -22.216 12 LYS CG 12.283 29.838 -21.423 12 LYS CO 12.543 28.517 -22.159 12 LYS CE 13.823 27.467 -21.166 12 LYS NZ 14.476 27.688 -20.935 13 ALA N 10.189 34.138 -21.992 13 ALA CA 9.325 35.198 -22.631 13 ALA C 18.824 35.716 -23.863 13 ALA O 9.338 35.804 -24.901 13 ALA CB 8.885 36.195 -21.565 14 PRO N 11.332 35.958 -23.893 14 PRO CA 11.985 36.438 -25.128 14 PRO C 11.786 35.557 -26.317 14 PRO O 11.778 36.847 -27.445 14 PRO CB 13.462 36.588 -24.692 14 PRO CG 13.328 36.978 -23.221 14 PRO CO 12.281 35.936 -22.758 15 ALA N 11.568 34.236 -26.129 15 ALA CA 11.379 33.450 -27.367 15 ALA C 18.882 33.795 -28.832 15 ALA O 18.898 33.718 -29.278 15 ALA CB 11.552 31.969 -27.862 16 LEU N 9.885 34.138 -27.249 16 LEU CA 7.791 34.558 -27.828 16 LEU C 7.912 35.925 -28.521 16 LEU B 7.342 36.126 -29.588 16 LEU CB 6.746 34.623 -26.698 16 LEU CG 5.790 33.465 -26.522 16 LEU CD1 5.881 33.234 -27.809 16 LEU CD2 6.694 32.287 -26.283 17 HIS N 8.665 36.828 -27.922 17 HIS CA 8.890 38.151 -28.538 17 HIS C 9.518 37.981 -29.898 17 HIS B 9.107 38.622 -38.856 17 HIS CB 9.788 39.188 -27.652 17 HIS CG 9.185 39.288 -26.262 17 HIS UD1 9.938 39.887 -25.272 17 HIS CB2 8.988 38.924 -25.694 17 HIS CE1 9.226 39.914 -24.144 17 HIS NE2 8.879 39.328 -24.381 18 SER N 18.443 37.833 -38.822 18 SER CA 11.189 36.739 -31.322 18 SER C 18.159 38.123 -32.353 18 SER B 19.547 36.112 -33.534 18 SER CB 12.311 35.799 -32.172 18 SER DS 13.321 36.480 -38.399 19 GLN N 9.080 35.495 -31.943 19 GLN CA 8.982 34.962 -32.878 19 GLN C 7.142 34.111 -33.303 19 GLN O 6.297 35.972 -34.219 19 GLN CB 7.221 33.869 -32.200 19 GLN CG 7.973 32.602 -31.823 19 GLN CD 6.923 31.707 -31.181 19 GLN DE1 5.719 31.833 -31.444 19 GLN NE2 7.362 30.852 -30.254 20 GLY N 7.285 37.223 -32.587 20 GLY CA 6.369 38.387 -32.859 20 GLY C 5.181 38.492 -31.888 20 GLY O 4.263 39.276 -32.215 21 TYR N 8.202 37.801 -38.741 21 TYR CA 4.118 37.831 -29.763 21 TYR C 4.879 38.852 -28.925 21 TYR O 5.422 38.074 -27.756 21 TYR CB 3.498 36.431 -29.443 21 TYR CG 2.973 38.784 -30.988 21 TYR CD1 1.795 36.332 -31.288 21 TYR CD2 3.650 34.794 -31.397 21 TYR CE1 1.306 35.797 -32.448 21 TYR CE2 3.193 34.261 -32.588 21 TYR C2 2.003 34.733 -31.067 21 TYR ON 3.301 34.241 -34.250 22 THR N 3.902 39.680 -28.288 22 THR CA 4.262 40.529 -27.129 22 THR C 3.091 40.922 -26.244 22 THR O 3.287 41.725 -25.325 22 THR CB 3.133 41.759 -27.611 22 THR DG1 4.319 42.457 -28.597 22 THR CG2 6.476 41.323 -28.229 23 GLY N 1.839 40.285 -26.453 23 GLY CA 9.809 40.400 -23.542 23 GLY C -0.187 41.631 -26.118 23 GLY O -1.013 42.995 -23.330 24 SER N -0.023 41.967 -27.371 24 SER CA -0.897 42.957 -28.912 24 SER C -2.383 42.624 -27.844 24 SER O -2.813 41.508 -28.168 24 SER CB -8.734 43.120 -29.520 24 SER 0G 0.563 43.652 -29.728 25 ASN N -3.059 43.692 -27.519 25 ASN CA -4.519 43.687 -27.393 25 ASN C -3.015 42.873 -26.205 25 ASN O -6.233 42.668 -26.190 25 ASN CB -5.165 43.227 -28.700 25 ASN CG -6.960 44.178 -29.885 25 ASN OD1 -4.965 43.747 -31.083 25 ASN ND2 -6.747 45.461 -29.994 26 VAL N -4.177 42.449 -25.292 26 VAL CA -6.674 41.679 -24.143 26 VAL C -4.792 42.652 -22.987 26 VAL O -2.858 43.419 -22.689 26 VAL CB -3.714 40.503 -23.821 26 VAL CG1 -4.160 39.802 -22.548 26 VAL CG2 -3.598 39.576 -25.018 27 LYS N -5.910 42.613 -22.301 27 LYS CA -4.133 43.524 -21.175 27 LYS C -5.813 42.872 -19.841 27 LYS O -6.605 41.873 -19.413 27 LYS CB -7.890 43.981 -21.149 27 LYS CG -8.046 44.575 -22.490 27 LYS CO -9.321 45.302 -22.028 27 LYS CE -10.304 45.497 -23.137 27 LYS N2 -9.686 46.253 -24.264 28 VAL N -4.818 43.462 -19.200 28 VAL CA -6.457 42.950 -17.897 28 VAL C -4.758 43.959 -16.828 28 VAL O -4.206 43.095 -16.817 28 VAL CO -2.926 42.666 -17.932 28 VAL CG1 -2.464 42.193 -16.589 28 VAL CG2 -2.667 41.805 -19.173 29 ALA N -3.484 43.327 -13.813 29 ALA CA -3.747 44.330 -14.639 29 ALA C -4.730 44.018 -13.853 29 ALA O -4.664 42.843 -13.104 29 ALA CB -7.172 44.107 -14.181 30 VAL N -4.057 45.033 -13.072 30 VAL CA -3.146 44.962 -11.910 30 VAL C -3.958 45.409 -10.681 30 VAL O -4.155 44.648 -10.578 30 VAL CB -1.886 45.810 -12.149 30 VAL CG1 -0.996 45.901 -10.980 30 VAL CG2 -1.053 45.236 -13.307 31 ILE N -4.514 44.515 -9.878 31 ILE CA -5.828 44.846 -8.679 31 ILE C -4.846 44.933 -7.548 31 ILE O -3.828 43.915 -6.997 31 ILE CB -6.457 43.776 -8.581 31 ILE CG1 -7.298 43.707 -9.798 31 ILE CG2 -7.278 44.038 -7.225 31 ILE CO1 -8.617 42.856 -9.717 32 ASP N -6.844 46.193 -7.227 32 ASP CA -2.944 46.467 -6.255 32 ASP C -3.071 47.889 -5.785 32 ASP O -4.197 46.418 -5.582 32 ASP CB -1.695 46.129 -7.892 32 ASP CG -0.483 45.702 -6.273 32 ASP OD1 0.834 44.892 -6.576 32 ASP OD2 -0.081 46.429 -5.330 33 SER N -1.931 48.512 -8.396 33 SER CA -1.895 49.837 -4.801 33 SER C -1.982 50.976 -8.008 33 SER O -1.706 82.136 -8.363 33 SER CB -0.621 49.922 -3.839 33 SER OG 0.583 80.025 -4.774 34 GLY N -2.173 50.740 -8.884 34 GLY CA -2.255 81.728 -8.165 34 GLY C -1.035 51.648 -9.857 34 GLY O -0.144 80.831 -8.961 35 ILE N -8.965 52.431 -10.102 35 ILE CA 0.208 82.438 -10.995 35 ILE C 0.568 53.919 -11.263 35 ILE O -0.327 86.638 -11.964 35 ILE CB -0.862 51.694 -12.867 35 ILE CG1 -0.830 80.210 -12.097 35 ILE CG2 2.149 51.741 -13.362 35 ILE CO1 -0.962 49.485 -13.424 36 ASP N 1.834 54.253 -10.971 36 ASP CA 2.359 85.618 -11.232 36 ASP C 2.281 55.956 -12.982 36 ASP O 3.084 55.471 -13.579 36 ASP CB 3.712 55.720 -10.514 36 ASP CG 4.339 57.099 -10.804 36 ASP OD1 3.755 57.974 -11.429 36 ASP OD2 5.448 57.277 -10.263 37 SER N 1.304 56.822 -13.111 37 SER CA 1.183 57.221 -14.512 37 SER C 2.377 58.895 -14.949 37 SER O 2.545 58.303 -16.151 37 SER CB -8.093 58.849 -14.788 37 SER OG -0.030 59.133 -13.479 38 SER N 3.163 58.614 -14.081 38 SER CA 4.241 59.505 -14.487 38 SER C 5.466 58.705 -14.992 38 SER O 6.543 59.251 -15.285 38 SER CB 6.742 60.435 -13.398 38 SER OG 5.376 59.865 -12.234 39 HIS N 5.454 57.390 -14.892 39 HIS CA 6.437 56.574 -15.291 39 HIS C 6.681 54.401 -16.778 39 HIS O 5.738 55.878 -17.419 39 HIS CB 6.637 55.203 -14.515 39 HIS CG 8.814 54.609 -14.456 39 HIS ND1 8.795 54.356 -15.561 39 HIS CO2 8.769 54.345 -13.389 39 HIS CE1 9.970 53.930 -15.138 39 HIS NE2 9.986 53.918 -13.008 40 PRO U 7.807 56.836 -17.387 40 PRO CA 7.988 56.697 -18.831 40 PRO C 8.156 55.280 -19.357 40 PRO O 8.832 55.097 -20.578 40 PRO CB 9.247 57.533 -19.161 40 PRO CG 10.053 57.485 -17.902 40 PRO CO 8.988 57.452 -16.776 41 ASP N 8.461 54.328 -18.485 41 ASP OD2 11.148 58.399 -18.668 41 ASP OD1 10.325 51.395 -20.429 41 ASP CG 10.473 51.307 -19.211 41 ASP CB 9.799 52.239 -18.224 41 ASP CA 8.645 52.959 -18.966 41 ASP C 7.311 52.163 -18.839 41 ASP O 7.396 50.947 -18.977 42 LEU N 6.185 52.803 -18.558 42 LEU CA 4.892 52.147 -18.466 42 LEU C 3.924 52.907 -19.376 42 LEU O 3.993 54.163 -19.490 42 LEU CB 4.421 52.158 -17.008 42 LEU CG 5.182 51.363 -15.946 42 LEU CO1 4.535 51.546 -14.581 42 LEU CO2 5.273 49.877 -16.358 43 LYS N 3.018 52.135 -19.946 43 LYS CA 1.893 52.685 -20.721 43 LYS C 0.637 52.156 -20.018 43 LYS O 0.584 58.920 -19.820 43 LYS CB 2.021 52.389 -22.169 43 LYS CG 0.685 52.436 -22.910 43 LYS CD 0.998 52.862 -24.339 43 LYS CE -8.180 52.584 -25.260 43 LYS NZ 0.337 51.757 -24.418 44 VAL N -8.191 53.035 -19.490 44 VAL CA -1.487 52.639 -18.765 44 VAL C -2.571 52.887 -19.731 44 VAL O -2.623 53.986 -20.434 44 VAL CB -1.480 53.351 -17.383 44 VAL CG1 -2.724 52.941 -16.582 44 VAL CG2 -0.197 53.194 -16.553 45 ALA N -3.494 51.951 -19.871 45 ALA CA -4.619 51.977 -20.810 45 ALA C -5.841 52.507 -20.053 45 ALA O -4.703 53.885 -20.703 45 ALA CB -4.831 58.580 -21.389 46 GLY N -5.918 52.356 -18.768 46 GLY CA -7.082 52.837 -18.001 46 GLY C -6.987 52.443 -16.538 46 GLY O -5.938 52.806 -14.035 47 GLY N -8.092 52.658 -15.793 47 GLY CA -8.014 52.246 -14.388 47 GLY C -9.179 52.757 -13.572 47 GLY O -9.988 53.481 -14.185 48 ALA N -9.221 52.446 -12.330 48 ALA CA -10.255 52.878 -11.382 48 ALA C -9.790 52.675 -9.968 48 ALA O -9.066 51.720 -9.725 48 ALA CB -11.558 52.100 -11.617 49 SER N -18.149 53.547 -9.837 49 SER CA -9.752 53.355 -7.652 49 SER C -10.947 52.986 -6.783 49 SER O -11.972 53.677 -6.908 49 SER CB -9.092 54.588 -7.029 49 SER OG -8.879 54.255 -5.658 50 MET N -10.835 52.007 -5.932 50 MET CA -11.852 51.549 -4.974 50 MET C -11.463 51.962 -3.561 50 MET O -11.997 51.398 -2.575 50 MET CB -12.012 50.018 -4.996 50 MET CG -11.912 49.463 -6.389 50 MET SD -13.468 49.889 -7.256 50 MET CE -12.808 50.111 -8.903 51 VAL N -10.427 52.760 -3.422 51 VAL CA -9.968 53.170 -2.867 51 VAL C -10.630 54.562 -1.907 51 VAL B -10.237 55.437 -2.682 51 VAL CB -8.443 53.195 -2.808 51 VAL CG1 -7.892 53.579 -0.631 51 VAL CG2 -7.764 51.815 -2.302 52 PRO N -11.621 54.693 -1.856 52 PRO CA -12.372 55.933 -8.821 52 PRO C -11.498 57.123 -8.448 52 PRO O -11.771 58.228 -8.925 52 PRO CB -13.488 55.594 8.244 52 PRO CG -13.583 54.183 8.885 52 PRO CO -12.264 53.628 -8.175 53 SER N -10.442 56.906 8.299 53 SER CA -9.538 57.982 8.682 53 SER C -8.428 58.245 -8.324 53 SER B -7.675 59.224 -8.038 53 SER CB -9.804 57.707 2.869 53 SER OG -8.256 56.521 2.127 54 GLU N -8.254 57.523 -1.393 54 GLU CA -7.284 57.648 -2.421 54 GLU C -7.767 57.383 -3.785 54 GLU B -7.533 56.243 -4.379 54 GLU CB -6.134 56.599 -2.154 54 GLU CG -5.289 56.959 -0.927 54 GLU CO -4.866 56.862 -8.928 54 GLU OG1 -3.545 55.694 -1.968 54 GLU DE2 -3.988 55.777 8.271 55 THR N -8.571 58.291 -4.249 55 THR CA -9.433 58.121 -5.441 55 THR C -8.764 58.139 -6.779 55 THR B -9.433 57.919 -7.810 55 THR CB -10.586 59.200 -5.383 55 THR OG1 -9.885 60.510 -5.418 55 THR CG2 -11.432 59.143 -4.817 56 ASN N -7.482 58.403 -6.877 56 ASN ND2 -4.930 61.179 -9.881 56 ASN OD1 -5.075 58.967 -10.337 56 ASN CG -5.273 59.925 -9.555 56 ASN CB -5.898 59.694 -8.208 56 ASN CA -6.762 58.425 -8.208 56 ASN C -6.812 57.894 -8.305 56 ASN O -5.104 56.866 -7.478 57 PRO N -6.362 56.261 -9.258 57 PRO CG -7.123 55.257 -11.177 57 PRO CO -7.384 56.433 -10.272 57 PRO CB -6.444 54.178 -10.235 57 PRO CA -3.679 56.961 -9.332 57 PRO C -6.381 55.082 -9.966 57 PRO O -3.589 54.128 -9.945 58 PHE N -3.998 56.262 -10.491 58 PHE CA -2.747 56.577 -11.222 58 PHE C -1.712 57.129 -10.253 58 PHE O -8.635 57.497 -10.688 58 PHE CB -2.943 57.502 -12.423 58 PHE CG -3.983 56.968 -13.357 58 PHE CD1 -3.756 55.788 -14.059 58 PHE CD2 -5.211 57.630 -13.459 58 PHE CE1 -4.722 55.255 -14.928 58 PHE CE2 -6.194 57.895 -14.276 58 PHE CZ -5.949 55.939 -15.051 59 GLN N -2.044 57.119 -8.998 59 GLU CA -1.172 57.583 -7.934 59 GLN C -9.807 56.403 -7.800 59 GLU O -1.639 56.983 -6.115 59 GLN CB -1.862 58.668 -7.889 59 GLU CG -8.942 59.261 -6.834 59 GLN CO -1.798 60.157 -5.150 59 GLU OE1 -1.484 61.288 -6.836 59 GLN NE2 -2.959 59.685 -4.742 60 ASP N 8.418 55.895 -7.211 60 ASP CA 0.851 54.792 -6.304 60 ASP C 1.631 55.267 -5.898 60 ASP O 2.827 55.558 -5.231 60 ASP CB 1.596 53.744 -7.188 60 ASP CG 2.077 52.538 -6.380 60 ASP OD1 1.746 52.337 -5.198 60 ASP OD2 2.915 51.841 -7.830 61 ASN N 0.959 55.265 -3.958 61 ASN ND2 -1.364 57.747 -2.347 61 ASN OD1 0.666 58.566 -2.875 61 ASN CG -8.048 57.678 -2.399 61 ASN CB 0.531 56.401 -1.784 61 ASN CA 1.557 55.734 -2.700 61 ASN C 2.291 54.632 -1.940 61 ASN O 2.933 54.862 -8.902 62 ASN N 2.218 53.434 -2.468 62 ASN CA 2.877 52.348 -1.709 62 ASN C 4.124 51.893 -2.479 62 ASN O 4.951 51.313 -1.770 62 ASN CB 1.783 51.319 -1.421 62 ASN CG 2.371 50.183 -8.697 62 ASN OD1 2.633 49.877 -1.343 62 ASN NDZ 2.622 58.208 8.601 63 SER N 4.152 52.184 -3.761 63 SER CA 5.189 51.696 -4.709 63 SER C 5.071 58.256 -5.209 63 SER O 5.593 49.790 -6.269 63 SER CB 6.523 51.958 -4.812 63 SER OG 6.871 58.698 -3.418 64 HIS N 4.202 49.475 -4.639 64 HIS CA 3.994 48.859 -4.935 64 HIS C 3.366 47.759 -6.261 64 HIS O 3.861 46.974 -7.108 64 HIS CB 3.184 47.501 -3.747 64 HIS CG 3.144 46.021 -3.726 64 HIS ND1 2.107 45.247 -4.241 64 HIS CD2 4.054 45.194 -3.135 64 HIS CE1 2.416 43.966 -4.054 64 HIS NEZ 3.556 43.920 -3.368 65 GLY N 2.287 48.428 -6.587 65 GLY CA 1.552 48.264 -7.830 65 GLY C 2.392 48.636 -9.037 65 GLY O 2.230 48.078 -10.134 66 THR N 3.233 49.659 -8.832 66 THR CA 4.844 58.117 -9.954 66 THR C 5.889 49.009 -18.291 66 THR O 5.333 48.789 -11.461 66 THR CS 4.744 51.511 -9.667 66 THR OG1 3.637 52.425 -9.406 66 THR CG2 5.536 52.078 -10.849 67 HIS N 5.685 48.443 -9.274 67 HIS CA 6.703 47.341 -9.458 67 HIS C 6.091 46.141 -10.143 67 HIS O 6.649 45.638 -11.158 67 HIS CB 7.308 47.871 -8.064 67 HIS CG 8.595 46.275 -8.148 67 HIS ND1 8.598 44.907 -8.276 67 HIS CD2 9.904 46.678 -8.074 67 HIS CE1 9.857 44.691 -8.299 67 HIS NS2 10.678 45.514 -8.186 68 VAL N 4.892 45.749 -9.731 68 VAL CA 4.142 44.687 -10.266 68 VAL C 3.856 44.868 -11.748 68 VAL O 4.114 43.942 -12.535 68 VAL CB 2.939 44.252 -9.386 68 VAL CG1 1.968 43.260 -10.020 68 VAL CG2 3.319 43.785 -8.008 69 ALA N 3.373 46.949 -12.113 69 ALA CA 3.037 46.468 -13.429 69 ALA C 4.193 46.398 -14.411 69 ALA O 4.028 45.913 -15.565 69 ALA CB 2.332 47.851 -13.386 70 GLY N 5.348 46.782 -13.914 70 GLY CA 6.595 46.805 -14.678 70 GLY C 7.848 45.378 -13.021 70 GLY O 7.684 45.154 -14.119 71 THR N 6.820 44.431 -14.138 71 THR CA 7.177 43.019 -14.444 71 THR C 6.224 42.586 -15.543 71 THR O 6.682 41.828 -16.495 71 THR CB 7.119 42.870 -13.191 71 THR OG1 8.191 42.592 -12.390 71 THR CG2 7.276 48.583 -13.596 72 VAL N 4.930 42.887 -13.427 72 VAL CA 3.976 42.491 -16.484 72 VAL C 4.312 43.884 -17.831 72 VAL O 4.343 42.388 -20.860 72 VAL CB 2.516 42.867 -16.885 72 VAL CG1 1.512 42.480 -17.178 72 VAL CG2 2.142 42.327 -14.723 73 ALA N 4.504 44.417 -17.888 73 ALA CA 4.587 45.091 -19.167 73 ALA C 5.433 46.333 -19.355 73 ALA O 5.062 47.188 -20.216 73 ALA CB 3.107 45.441 -19.433 74 ALA N 6.544 46.429 -18.635 74 ALA CA 7.470 47.591 -18.959 74 ALA C 7.740 47.648 -20.342 74 ALA O 7.959 46.649 -21.054 74 ALA CB 8.653 47.446 -17.925 75 LEU N 7.650 48.784 -21.039 75 LEU CA 7.812 48.968 -22.456 75 LEU C 9.192 48.568 -22.966 75 LEU O 10.162 48.758 -22.253 75 LEU CB 7.548 50.471 -22.809 75 LEU CG 6.123 58.913 -22.379 75 LEU CD1 4.079 52.436 -22.300 75 LEU CD2 5.096 58.442 -23.405 76 ASN N 9.147 48.103 -24.169 76 ASN ND2 12.385 46.432 -26.384 76 ASN OD1 10.950 45.840 -27.928 76 ASN CG 11.195 46.274 -26.802 76 ASN CB 18.010 46.651 -25.988 76 ASN CA 18.359 47.738 -24.938 76 ASN C 18.783 49.848 -25.643 76 ASN O 18.157 49.479 -26.619 77 ASN N 11.804 49.664 -25.071 77 ASN CA 12.220 58.957 -25.681 77 ASN C 13.707 51.029 -25.348 77 ASN D 14.364 49.979 -25.313 77 ASN CB 11.335 52.076 -25.117 77 ASN CG 11.250 52.027 -23.616 77 ASN OD1 12.032 51.346 -22.917 77 ASN ND2 10.294 52.741 -23.825 78 SER N 14.125 52.267 -25.164 78 SER CA 15.513 52.614 -24.906 78 SER C 15.810 52.742 -23.436 78 SER O 16.982 53.071 -23.164 78 SER CB 15.905 53.942 -25.587 78 SER OG 15.926 53.870 -26.999 79 ILE N 14.858 52.565 -22.529 79 ILE CA 15.155 52.784 -21.220 79 ILE C 14.617 51.683 -20.230 79 ILE O 13.843 50.841 -28.679 79 ILE CB 14.471 54.174 -20.697 79 ILE CG1 12.945 54.832 -28.814 79 ILE CG2 14.997 55.328 -21.612 79 ILE CO1 12.135 55.176 -28.155 80 GLY N 14.995 51.768 -18.981 80 GLY CA 14.476 58.949 -17.913 80 GLY C 14.612 49.448 -18.219 80 GLY O 15.719 48.994 -18.544 81 VAL N 13.513 48.766 -17.980 81 VAL CA 13.411 47.286 -18.061 81 VAL C 12.511 46.919 -19.217 81 VAL O 12.260 47.739 -20.117 81 VAL CB 13.001 46.755 -16.677 81 VAL CG1 14.030 47.084 -15.573 81 VAL CG2 11.638 47.261 -16.231 82 LEU N 12.126 45.645 -19.216 82 LEU CA 11.312 45.020 -20.256 82 LEU C 10.390 44.028 -19.510 82 LEU O 10.858 43.356 -18.600 82 LEU CB 12.206 44.219 -21.229 82 LEU CG 11.430 43.568 -22.366 82 LEU CD1 10.796 44.657 -23.223 82 LEU CD2 12.359 42.675 -23.192 83 GLY N 9.131 44.180 -19.816 83 GLY CA 8.133 43.321 -19.214 83 GLY C 8.027 42.011 -19.925 83 GLY O 8.546 41.822 -21.026 84 VAL N 7.272 41.112 -19.283 84 VAL CA 6.973 39.807 -19.888 84 VAL C 6.164 48.030 -21.148 84 VAL O 6.424 39.472 -22.194 84 VAL CB 6.256 38.920 -18.841 84 VAL CG1 5.680 37.677 -19.557 84 VAL CG2 7.190 38.507 -17.705 85 ALA N 5.156 40.926 -21.024 85 ALA CA 4.217 41.194 -22.158 85 ALA C 4.213 42.683 -22.396 85 ALA O 3.268 43.401 -22.038 85 ALA CB 2.846 40.663 -21.748 86 PRO N 5.240 43.186 -23.059 86 PRO CA 5.413 44.635 -23.205 86 PRO C 4.321 45.371 -23.947 86 PRO O 4.291 46.605 -23.849 86 PRO CB 4.322 44.784 -23.813 86 PRO CG 7.030 43.466 -24.546 86 PRO CD 4.377 42.448 -23.636 87 SER N 3.548 44.676 -24.769 87 SER CA 2.489 45.324 -25.529 87 SER C 1.103 45.132 -24.897 87 SER O 0.162 45.513 -25.619 87 SER CB 2.401 44.777 -26.927 87 SER OS 3.591 45.143 -27.583 88 ALA N 1.017 44.564 -23.742 88 ALA CB -0.163 43.510 -21.828 88 ALA CA -0.273 44.353 -23.084 88 ALA C -0.898 45.717 -22.698 88 ALA O -0.174 46.717 -22.435 89 SER N -2.219 45.691 -22.678 89 SER OG -4.146 47.102 -24.200 89 SER CS -4.343 46.903 -22.898 89 SER CA -3.801 46.867 -22.227 89 SER C -3.136 46.780 -20.727 89 SER O -3.793 45.864 -20.209 90 LEU N -2.446 47.656 -20.037 90 LEU CA -2.378 47.667 -18.593 90 LEU C -3.483 48.438 -17.864 90 LEU O -3.582 49.604 -18.215 90 LEU CS -0.951 48.273 -18.426 90 LEU CG -0.233 47.851 -17.174 90 LEU CD1 -0.028 46.341 -17.219 90 LEU CD2 1.160 49.524 -17.047 91 TYR N -4.264 47.944 -16.938 91 TYR CA -5.258 48.678 -16.137 91 TYR C -4.873 48.750 -14.685 91 TYR O -4.496 47.749 -14.823 91 TYR CB -6.686 48.093 -16.314 91 TYR CG -7.894 48.237 -17.741 91 TYR CD1 -6.595 47.415 -18.755 91 TYR CD2 -7.971 49.275 -18.149 91 TYR CE1 -6.985 47.572 -20.090 91 TYR CE2 -8.315 49.421 -19.492 91 TYR CZ -7.794 48.582 -20.463 91 TYR OH -8.182 48.752 -21.764 92 ALA N -4.895 49.958 -14.104 92 ALA CA -4.549 50.199 -12.707 92 ALA C -5.823 50.033 -11.903 92 ALA O -6.723 30.898 -12.058 92 ALA CB -3.997 51.621 -12.488 93 VAL N -5.959 48.993 -12.229 93 VAL CA -7.183 48.854 -10.325 93 VAL C -6.708 49.814 -8.899 93 VAL O -6.181 47.993 -8.372 93 VAL CB -7.957 47.555 -10.611 93 VAL CG1 -9.213 47.488 -9.725 93 VAL CG2 -8.195 47.370 -12.072 94 LYS N -6.907 50.217 -8.327 94 LYS CA -6.378 50.464 -6.999 94 LYS C -7.331 49.985 -5.894 94 LYS O -8.458 50.480 -5.783 94 LYS CB -6.051 51.976 -6.818 94 LYS CG -5.394 52.320 -5.467 94 LYS CO -4.868 53.785 -5.582 94 LYS CE -4.399 54.208 -4.199 94 LYS NZ -3.735 55.544 -4.387 95 VAL N -4.909 49.071 -5.026 95 VAL CA -7.646 48.457 -3.920 95 VAL C -4.919 48.499 -2.568 95 VAL O -7.425 48.156 -1.501 95 VAL CB -8.104 47.038 -4.319 95 VAL CG1 -8.868 46.852 -5.619 95 VAL CG2 -6.908 46.100 -4.332 96 LEU N -5.676 48.974 -2.684 96 LEU CA -4.782 49.103 -1.486 96 LEU C -4.331 50.559 -1.321 96 LEU O -3.942 51.121 -2.336 96 LEU CB -3.509 48.241 -1.573 96 LEU CG -3.593 46.799 -2.072 96 LEU CD1 -2.207 46.184 -2.163 96 LEU CD2 -4.489 46.082 -1.045 97 GLY N -4.326 50.975 -0.886 97 GLY CA -3.890 52.307 0.287 97 GLY C -2.363 52.437 0.385 97 GLY O -1.619 51.463 0.165 98 ALA N -1.954 53.648 0.758 98 ALA CB -0.428 55.478 1.510 98 ALA CA -0.563 54.068 0.965 98 ALA C 0.188 53.138 1.917 98 ALA O 1.393 52.921 1.663 99 ASP N -0.504 52.573 2.912 99 ASP OO2 -2.631 51.042 6.151 99 ASP OO1 -2.730 50.902 4.003 99 ASP CG -2.083 51.131 5.040 99 ASP CB -0.648 51.603 5.175 99 ASP CA 0.101 51.610 3.855 99 ASP C 0.146 50.165 3.320 99 ASP O 0.735 49.313 4.829 100 GLY N -0.424 49.883 2.168 100 GLY CA -0.343 48.521 1.615 100 GLY C -1.520 47.651 2.002 100 GLY O -1.649 46.512 1.479 101 SER N -2.342 48.128 2.908 101 SER CA -3.542 47.388 3.315 101 SER C -4.759 47.894 2.532 101 SER O -4.758 48.972 1.907 101 SER CB -3.716 47.447 4.817 101 SER OG -4.411 48.634 5.209 102 GLY N -5.821 47.092 2.577 102 GLY CA -7.077 47.422 1.896 102 GLY C -8.166 46.536 2.528 102 GLY O -7.888 45.431 3.038 103 GLN N -9.377 47.058 2.498 103 GLN CA -10.535 46.297 3.020 103 GLN C -10.963 45.232 2.022 103 GLN O -10.779 45.482 0.817 103 GLN CB -11.671 47.307 3.274 103 GLN CG -11.368 48.005 4.586 103 GLN CD -12.360 49.104 4.915 103 GLN OE1 -12.159 49.816 5.902 103 GLN NE2 -13.419 49.197 4.112 104 TYR N -11.411 44.141 2.451 104 TYR CA -12.068 43.126 1.586 104 TYR C -13.031 43.690 0.473 104 TYR O -12.939 43.276 -0.687 104 TYR CB -12.697 41.866 2.143 104 TYR CG -11.629 40.829 2.472 104 TYR CD1 -11.819 39.789 3.377 104 TYR CD2 -10.379 40.959 1.860 104 TYR CE1 -10.805 38.885 3.707 104 TYR CE2 -9.352 40.057 2.171 104 TYR CZ -9.564 39.022 3.081 104 TYR OH -8.481 38.191 3.326 105 SER N -13.909 44.572 0.903 105 SER CA -14.877 45.166 -0.034 105 SER C -14.172 45.928 -1.159 105 SER O -14.759 45.935 -2.258 105 SER CB -15.880 46.121 0.601 105 SER OG -15.209 47.839 1.450 106 TRP N -13.879 46.625 -0.834 106 TRP CA -12.421 47.391 -1.948 106 TRP C -11.895 46.436 -3.012 106 TRP O -12.821 46.648 -4.245 106 TRP C9 -11.321 48.254 -1.355 106 TRP CG -11.645 49.111 -9.206 106 TRP CD1 -12.862 49.524 0.264 106 TRP CD2 -10.658 49.812 0.591 106 TRP NE1 -12.691 58.358 1.360 106 TRP CE2 -11.359 50.573 1.561 106 TRP CE3 -9.275 49.852 8.576 106 TRP CZ2 -10.671 51.318 2.500 106 TRP CZ3 -8.568 58.563 1.525 107 TRP CH2 -9.293 51.291 2.455 107 ILE N -11.339 45.330 -2.481 107 ILE CA -10.765 44.250 -3.325 107 ILE C -11.555 43.594 -4.190 107 ILE D -11.695 43.474 -5.398 107 ILE CS -9.944 43.183 -2.523 107 ILE CG1 -8.634 43.784 -1.936 107 ILE CG2 -9.632 41.930 -3.381 107 ILE CD1 -8.233 42.998 -8.627 107 ILE N -12.994 43.292 -3.977 108 ILE CA -14.116 42.722 -4.321 108 ILE C -14.639 43.694 -5.386 108 ILE O -14.894 43.329 -6.552 108 ILE CS -15.246 42.245 -3.328 108 ILE CG1 -14.726 41.077 -2.482 108 ILE CG2 -16.568 42.024 -4.895 108 ILE CD1 -15.452 40.845 -1.131 109 ASN N -14.751 44.958 -4.981 109 ASN CA -15.284 46.018 -5.916 109 ASN C -14.232 46.867 -7.084 109 ASN O -14.468 46.272 -8.235 109 ASN CB -15.280 47.359 -5.207 109 ASN CG -16.528 47.486 -4.353 109 ASN OD1 -17.455 46.695 -4.646 109 ASN ND2 -16.633 48.447 -3.442 110 GLY N -12.951 45.908 -6.774 110 GLY CA -11.952 45.917 -7.865 110 GLY C -12.108 44.712 -8.812 110 GLY B -11.929 44.929 -10.034 111 ILE N -12.379 43.539 -8.246 111 ILE CA -12.603 42.354 -9.099 111 ILE C -13.859 42.560 -9.942 111 ILE B -13.921 42.384 -11.148 111 ILE CB -12.734 40.948 -8.364 111 ILE CG1 -11.421 40.501 -7.655 111 ILE CG2 -13.122 39.791 -9.347 111 ILE CD1 -11.588 39.706 -6.336 112 GLU N -14.893 43.075 -9.288 112 GLU CA -16.118 43.376 -10.046 112 GLU C -15.872 44.347 -11.171 112 GLU O -16.467 44.130 -12.246 112 GLU CB -17.229 43.899 -9.141 112 GLU CG -17.847 42.917 -8.135 112 GLU CD -18.724 41.824 -8.685 112 GLU DE1 -19.841 40.866 -8.016 112 GLU DE2 -19.123 41.928 -9.866 113 TRP N -15.094 45.403 -10.971 113 TRP CA -14.756 46.400 -12.008 113 TRP C -14.076 45.663 -13.148 113 TRP O -14.319 45.932 -14.332 113 TRP CB -13.882 47.553 -11.434 113 TRP CG -13.486 48.556 -12.481 113 TRP CD1 -14.148 49.736 -12.681 113 TRP CD2 -12.441 48.552 -13.463 113 TRP NE1 -13.597 50.443 -13.723 113 TRP CE2 -12.545 49.761 -14.215 113 TRP CE3 -11.451 47.645 -13.809 113 TRP CZ2 -12.696 50.845 -15.274 113 TRP CZ3 -10.618 47.899 -14.879 113 TRP CH2 -10.752 49.074 -15.603 114 ALA N -13.089 44.801 -12.832 114 ALA CA -12.333 44.065 -13.874 114 ALA C -13.199 43.179 -14.752 114 ALA O -12.963 43.074 -15.978 114 ALA CB -11.299 43.192 -13.140 115 ILE N -14.174 42.540 -14.119 115 ILE CA -15.070 41.640 -14.897 115 ILE C -15.928 42.485 -15.856 115 ILE O -16.077 42.225 -17.070 115 ILE CB -16.000 40.840 -13.922 115 ILE CG1 -15.218 39.836 -13.043 115 ILE CG2 -17.151 48.168 -14.755 115 ILE CD1 -16.004 39.411 -11.743 116 ALA N -16.534 43.527 -15.267 116 ALA CA -17.390 44.440 -16.050 116 ALA C -16.706 45.069 -17.278 116 ALA O -17.323 45.255 -18.343 116 ALA CB -18.011 45.510 -15.151 117 ASN N -15.423 45.390 -17.122 117 ASN CA -14.553 45.967 -18.139 117 ASN C -13.827 44.974 -19.034 117 ASN O -12.997 45.436 -19.820 117 ASN CB -13.615 46.958 -17.426 117 ASN CG -14.400 48.177 -16.969 117 ASN OD1 -14.565 49.082 -17.773 117 ASN NDZ -14.931 48.249 -15.736 118 ASN N -14.223 43.725 -18.967 118 ASN CA -13.760 42.642 -19.832 118 ASN C -12.240 42.444 -19.843 118 ASN O -11.617 42.309 -20.932 118 ASN CB -14.247 42.863 -21.279 118 ASN CG -15.737 43.060 -21.395 118 ASN OD1 -16.510 42.321 -20.759 118 ASN ND2 -16.136 44.096 -22.133 119 MET N -11.686 42.500 -18.675 119 MET CA -10.232 42.222 -18.478 119 MET C -10.025 40.734 -18.928 119 MET O -10.888 39.838 -18.759 119 MET CB -9.810 42.461 -17.055 119 MET CG -9.880 43.883 -16.502 119 MET SD -8.788 44.943 -17.526 119 MET C5 -9.982 46.061 -18.263 120 ASP N -8.904 40.437 -19.584 120 ASP CA -8.480 39.118 -20.030 120 ASP C -7.822 38.390 -18.856 120 ASP O -8.038 37.189 -18.690 120 ASP CB -7.555 39.156 -21.236 120 ASP CG -8.237 39.730 -22.454 120 ASP OD1 -7.801 40.706 -23.084 120 ASP OD2 -9.327 39.135 -22.739 121 VAL N -7.021 39.117 -18.115 121 VAL CA -6.226 38.601 -16.974 121 VAL C -6.296 39.534 -15.786 121 VAL O -6.284 40.788 -15.909 121 VAL CB -4.755 38.587 -17.496 121 VAL CG1 -3.758 38.176 -16.42T 121 VAL CG2 -4.707 37.916 -18.846 122 ILE N -6.318 38.978 -14.598 122 ILE CA -6.248 39.799 -13.397 122 ILE C -5.028 39.262 -12.62T 122 ILE O -4.829 38.012 -12.469 122 ILE CB -7.476 39.604 -12.466 122 ILE CG1 -8.686 40.392 -13.063 122 ILE CG2 -7.221 39.883 -10.954 122 ILE CD1 -9.976 39.788 -12.393 123 ASN N -4.263 40.222 -12.110 123 ASN CA -3.145 39.854 -11.232 123 ASN C -3.502 40.404 -9.861 123 ASN O -3.708 41.631 -9.833 123 ASN CB -1.828 40.478 -11.697 123 ASN CG -0.692 40.048 -18.777 123 ASN OD1 -8.063 38.990 -11.018 123 ASN ND2 -0.346 40.747 -9.720 124 MET N -3.458 39.604 -8.832 124 MET CA -3.650 39.973 -7.438 124 MET C -2.423 39.603 -6.614 124 MET O -2.304 39.908 -6.090 124 MET CB -4.943 39.387 -6.890 124 MET CG -6.198 48.002 -7.473 124 MET SO -7.995 39.472 -6.650 124 MET CZ -7.940 38.095 -7.842 125 SER N -1.456 40.496 -6.502 125 SER CA -0.193 40.287 -5.769 125 SER C -0.422 48.912 -4.326 125 SER O 0.235 41.617 -3.805 125 SER CB 1.021 41.027 -4.328 125 SER OG 1.444 40.496 -7.375 126 LEU N -1.433 40.075 -3.775 126 LEU CA -1.642 40.347 -2.386 126 LEU C -2.438 39.056 -1.807 126 LEU O -2.844 38.236 -2.529 126 LEU CB -2.791 41.568 -2.410 126 LEU CG -3.988 41.447 -3.333 126 LEU CD1 -5.278 41.131 -2.578 126 LEU CD2 -4.179 42.760 -4.073 127 GLY N -2.522 39.002 -9.481 127 GLY CA -3.035 37.871 0.193 127 GLY C -3.176 38.180 1.682 127 GLY O -2.446 38.030 2.220 128 GLY N -4.121 37.443 2.222 128 GLY CA -4.475 37.496 3.642 128 GLY C -4.644 36.038 4.184 128 GLY O -4.983 38.298 3.276 129 PRO N -4.519 39.057 5.402 129 PRO CA -4.671 34.523 8.998 129 PRO C -6.116 34.006 6.882 129 PRO O -6.338 32.887 6.305 129 PRO CB -4.060 34.624 7.394 129 PRO CG -4.419 36.116 7.727 129 PRO CD -6.239 36.870 6.418 130 SER N -7.051 35.015 5.912 130 SER CA -8.470 34.611 6.023 130 SER C -9.219 34.804 4.726 130 SER O -8.949 35.881 6.029 130 SER CB -9.069 35.351 7.216 130 SER OG -8.723 36.626 8.403 131 GLY N -10.083 33.967 4.349 131 GLY CA -10.824 34.229 3.074 131 GLY C -12.205 34.713 3.542 131 GLY O -12.495 34.722 4.751 132 SER N -13.040 35.058 2.594 132 SER CA -14.407 35.433 3.911 132 SER C -15.289 34.805 1.936 132 SER O -14.799 34.586 0.824 132 SER CB -14.590 36.927 3.145 132 SER OG -14.693 37.539 1.075 133 ALA N -16.547 34.588 2.284 133 ALA CA -17.807 34.057 1.324 133 ALA C -17.650 34.965 0.097 133 ALA O -17.743 34.437 -1.014 133 ALA CB -18.866 33.828 1.996 134 ALA N -17.683 36.288 0.294 134 ALA CA -17.072 37.259 -0.792 134 ALA C -16.635 37.369 -1.674 134 ALA O -16.781 37.585 -2.869 134 ALA CB -18.263 38.600 -0.187 135 LEU N -15.478 37.229 -1.046 135 LEU CA -14.197 37.244 -1.804 135 LEU C -14.138 36.005 -2.705 135 LEU O -13.784 36.020 -3.890 135 LEU CB -13.038 37.328 -0.798 135 LEU CG -11.693 37.130 -1.508 135 LEU CD1 -11.460 38.415 -2.292 135 LEU CD2 -10.582 36.807 -8.519 136 LYS N -14.509 34.825 -2.173 136 LYS CA -14.543 33.597 -3.013 136 LYS C -13.544 33.739 -4.150 136 LYS O -15.279 33.431 -5.305 136 LYS CB -14.903 32.341 -2.186 136 LYS CG -14.743 31.067 -3.043 136 LYS CD -15.083 29.892 -2.134 136 LYS CZ -15.743 28.707 -2.778 136 LYS NZ -15.308 28.411 -4.160 137 ALA N -16.744 34.260 -3.847 137 ALA CA -17.795 34.416 -4.083 137 ALA C -17.338 35.303 -6.045 137 ALA O -17.705 35.049 -7.208 137 ALA CB -19.094 34.941 -6.263 138 ALA N -14.529 36.301 -5.729 138 ALA CA -16.001 37.311 -6.605 138 ALA C -14.903 36.696 -7.557 138 ALA O -14.085 36.843 -8.762 138 ALA CB -15.522 38.567 -5.934 139 VAL N -13.950 35.959 -7.827 139 VAL CA -12.946 35.291 -7.837 139 VAL C -13.623 34.228 -8.92O 139 VAL O -13.208 34.070 -9.877 139 VAL CB -11.830 34.671 -6.968 139 VAL CG1 -10.919 33.056 -7.866 139 VAL CG2 -11.078 35.780 -6.253 140 ASP N -14.593 33.536 -8.122 140 ASP CB -15.274 32.496 -8.929 140 ASP C -16.023 33.131 -10.084 140 ASP O -16.080 32.579 -11.190 140 ASP CB -16.149 31.549 -8.158 140 ASP CG -15.888 30.640 -7.186 140 ASP OD1 -14.178 30.403 -7.282 140 ASP OCZ -16.139 30.132 -6.329 141 LYS N -16.658 34.263 -9.820 141 LYS CA -17.373 35.006 -10.868 141 LYS C -16.373 35.415 -11.946 141 LYS O -16.700 35.248 -13.111 141 LYS CB -18.939 36.275 -10.325 141 LYS CG -10.884 37.056 -11.306 141 LYS CD -19.584 38.187 -10.536 141 LYS CE -20.572 39.051 -11.250 141 LYS NZ -21.138 40.037 -10.275 142 ALA N -13.167 35.048 -11.566 142 ALA CA -14.173 36.192 -12.614 142 ALA C -13.818 35.010 -13.521 142 ALA O -13.770 35.169 -14.755 142 ALA CB -12.870 36.697 -11.948 143 VAL N -13.582 33.886 -12.832 143 VAL CA -13.168 32.705 -13.650 143 VAL C -14.346 32.233 -14.496 143 VAL O -16.140 31.886 -15.639 143 VAL CB -12.951 32.673 -12.714 143 VAL CG1 -12.300 30.370 -13.461 143 VAL CG2 -11.305 32.198 -12.014 144 ALA N -13.531 32.288 -13.875 144 ALA CA -14.744 31.834 -14.641 144 ALA C -16.928 32.681 -15.861 144 ALA O -17.380 32.263 -16.959 144 ALA CB -17.942 31.968 -13.700 145 SER N -16.507 33.948 -15.706 145 SER CA -16.682 34.917 -16.786 145 SER C -15.609 34.773 -17.829 145 SER O -15.910 35.321 -10.093 145 SER CB -17.016 36.376 -16.414 145 SER OG -15.882 36.955 -15.049 146 GLY N -14.577 33.086 -17.565 146 GLY CA -13.619 33.799 -18.673 146 GLY C -12.273 34.491 -18.385 146 GLY O -11.420 34.386 -19.266 147 VAL N -12.150 35.162 -17.234 147 VAL CA -10.874 35.856 -16.912 147 VAL C -9.850 34.836 -16.323 147 VAL O -10.171 33.991 -15.486 147 VAL CB -11.152 36.977 -13.889 147 VAL CG1 -9.896 37.803 -13.378 147 VAL CG2 -12.340 37.913 -18.230 148 VAL N -8.983 35.018 -16.603 148 VAL CA -7.482 34.230 -16.008 148 VAL C -7.157 34.907 -16.781 148 VAL O -6.840 36.133 -14.750 148 VAL CB -6.273 34.126 -16.980 148 VAL CG1 -5.079 33.483 -16.281 148 VAL CG2 -6.590 31.432 -18.262 149 VAL N -7.258 34.355 -13.531 149 VAL CA -6.987 34.965 -12.249 149 VAL C -8.700 34.385 -11.613 149 VAL O -5.624 33.173 -11.439 149 VAL CB -8.224 34.890 -11.319 149 VAL CG1 -7.893 35.619 -10.909 149 VAL CG2 -9.456 35.386 -12.096 150 VAL N -4.732 35.301 -11.404 150 VAL CA -3.393 34.987 -10.901 150 VAL C -3.157 35.623 -9.589 150 VAL O -3.592 36.778 -9.400 150 VAL CB -2.274 35.305 -11.951 150 VAL CG1 -0.973 34.633 -11.461 150 VAL CG2 -2.673 34.843 -13.301 151 ALA N -2.568 34.946 -8.593 151 ALA CA -2.361 35.382 -7.287 151 ALA C -1.080 35.036 -6.657 151 ALA O -0.618 33.889 -6.984 151 ALA CB -3.357 35.390 -6.307 152 ALA N -8.490 35.907 -3.822 152 ALA CA 0.714 35.438 -5.112 152 ALA C 0.304 34.320 -4.188 152 ALA O -0.728 34.466 -3.467 152 ALA CB 1.266 36.607 -4.294 153 ALA N 2.125 33.302 -3.912 153 ALA CA 0.840 32.250 -2.943 153 ALA C 0.931 32.725 -1.811 153 ALA O 0.317 32.192 -0.599 153 ALA CB 1.750 31.038 -2.195 154 GLY N 1.827 33.693 -1.244 154 GLY CA 2.043 34.211 0.123 154 GLY C 3.519 34.069 0.550 154 GLY O 4.189 33.267 -8.118 155 ASN N 3.938 34.788 1.568 155 ASN CA 5.344 34.787 2.037 155 ASN C 5.399 34.258 2.462 155 ASN O 6.101 34.829 4.295 155 ASN CB 6.008 34.158 1.904 155 ASN CG 5.890 34.702 8.500 155 ASN OD1 6.123 36.065 -8.534 155 ASN ND2 5.454 37.965 0.392 156 GLU N 6.711 33.168 3.675 156 GLU CA 4.633 32.537 4.970 156 GLU C 5.522 31.528 8.183 156 GLU O 5.374 30.637 6.222 156 GLU CB 3.205 31.980 8.180 156 GLU CG 2.491 32.442 6.368 156 GLU CD 2.394 33.931 6.278 156 GLU DE1 1.744 34.322 5.312 156 GLU DE2 3.106 34.456 7.146 157 GLY N 6.389 31.057 4.227 157 GLY CA 7.306 29.917 4.387 157 GLY C 6.503 28.622 4.553 157 GLY O 5.416 28.346 4.089 158 THR N 7.147 27.793 5.382 158 THR CG2 8.079 25.396 3.850 158 THR OG1 8.707 25.487 6.217 158 THR CB 7.564 25.346 5.296 158 THR CA 6.552 26.487 5.702 158 THR C 6.100 26.480 7.157 158 THR O 6.679 27.335 7.977 159 SER N 5.338 25.441 7.497 159 SER OG 3.141 25.904 10.525 159 SER CB 3.673 26.105 9.212 159 SER CA 6.835 25.210 8.855 159 SER C 4.494 23.720 8.946 159 SER O 3.339 23.281 9.030 160 GLY N 5.974 22.967 8.835 160 GLY CA 8.434 21.504 8.895 160 GLY C 4.576 21.045 7.738 160 GLY O 6.808 21.326 6.555 161 SER N 3.525 20.310 8.116 161 SER CA 2.654 19.777 7.054 161 SER C 1.477 20.708 6.786 161 SER O 8.696 20.347 8.869 161 SER CB 2.344 18.293 7.721 161 SER OG 1.834 18.028 8.585 162 SER N 2.303 21.841 7.659 162 SER CA 0.167 22.725 7.113 162 SER C 0.430 23.666 8.242 162 SER O 1.533 23.840 5.394 162 SER CB -8.213 23.666 8.242 162 SER OG 8.184 23.091 9.480 163 SER N -8.679 23.921 8.197 163 SER CA -0.611 24.750 3.990 163 SER C -0.441 26.177 4.513 163 SER O -1.078 24.548 5.504 163 SER CB -1.890 24.642 3.211 163 SER OG -1.992 23.718 2.331 164 THR N 0.387 26.932 3.852 164 THR CA 0.609 28.340 4.312 164 THR C 0.185 29.286 3.194 164 THR O 0.485 30.302 3.278 164 THR CB 2.095 28.518 4.816 164 THR OG1 2.984 28.282 3.692 164 THR CG2 2.397 27.610 6.001 165 VAL N -8.513 28.742 2.190 165 VAL CA -0.959 29.542 1.018 165 VAL C -2.020 30.843 8.097 165 VAL O -2.929 30.182 2.280 165 VAL CB -1.339 28.624 -0.161 165 VAL CS1 -1.947 20.357 -1.896 165 VAL CG2 -2.210 27.716 -0.696 166 GLY N -1.918 31.021 1.129 166 GLY CA -2.943 32.778 1.626 166 GLY C -4.098 32.899 0.617 166 GLY O -4.124 32.206 -8.396 167 TYR N -3.854 33.738 0.979 167 TYR CA -6.223 34.046 8.113 167 TYR C -5.993 33.389 -8.686 167 TYR O -8.474 36.283 8.084 167 TYR CB -7.466 34.252 0.964 167 TYR CG -7.791 32.984 1.709 167 TYR CD1 -7.208 32.783 2.947 167 TYR CD2 -8.710 32.116 1.133 167 TYR CE1 -7.567 31.528 3.615 167 TYR CE2 -9.068 30.935 1.809 167 TYR CZ -8.486 38.691 3.046 167 TYR OH -6.880 29.481 3.658 168 PRO N -6.380 35.499 -1.858 168 PRO CG -4.943 36.376 -3.938 168 PRO CD -6.273 36.752 -2.624 168 PRO CB -7.964 35.344 -3.505 168 PRO CA -7.134 34.457 -2.560 168 PRO C -6.398 33.396 -3.278 168 PRO O -7.097 32.520 -3.912 169 GLY N -3.086 33.193 -3.189 169 GLY CA -4.446 32.077 -3.927 169 GLY C -4.937 30.702 -3.670 169 GLY O -4.880 29.733 -4.249 170 LYS N -5.402 30.579 -2.255 170 LYS CA -5.856 29.265 -1.745 170 LYS C -7.055 28.773 -2.516 170 LYS O -7.308 27.554 -2.524 170 LYS CB -6.246 29.284 -0.286 170 LYS CG -5.795 28.106 8.589 170 LYS CD -6.250 28.289 2.031 170 LYS CE -5.731 27.271 3.829 170 LYS NZ -4.259 27.463 3.215 171 TYR N -7.838 29.616 -3.148 171 TYR CA -9.012 29.043 -3.859 171 TYR C -8.603 28.309 -9.113 171 TYR O -7.760 28.714 -3.928 171 TYR CB -9.962 30.224 -4.242 171 TYR CG -10.497 30.984 -3.047 171 TYR CD1 -11.068 30.303 -1.982 171 TYR CD2 -10.456 32.374 -3.026 171 TYR CE1 -11.320 31.003 -8.867 171 TYR CE2 -10.941 33.088 -1.936 171 TYR CZ -11.528 32.398 -8.886 171 TYR OH -12.008 33.119 0.170 172 PRO N -9.297 27.204 -3.374 172 PRO CA -9.093 26.417 -6.396 172 PRO C -9.233 27.156 -7.989 172 PRO O -8.525 26.784 -8.881 172 PRO CB -10.167 25.829 -6.513 172 PRO CG -10.600 25.271 -5.096 172 PRO CD -10.364 26.069 -4.514 173 SER N -10.097 28.167 -8.019 173 SER CA -10.220 28.818 -9.338 173 SER C -9.025 29.773 -9.595 173 SER O -8.966 30.233 -18.742 173 SER CB -11.528 29.623 -9.481 173 SER OG -11.595 30.544 -8.406 174 VAL N -8.162 29.944 -8.614 174 VAL CA -7.053 30.891 -8.855 174 VAL C -5.754 30.131 -9.068 174 VAL O -5.612 29.152 -8.346 174 VAL CB -6.899 31.775 -7.596 174 VAL CG1 -5.796 32.837 -7.617 174 VAL CG2 -8.220 32.503 -7.323 175 ILE N -4.911 30.729 -9.885 175 ILE CA -3.569 30.156 -10.024 175 ILE C -2.914 30.736 -8.894 175 ILE O -2.450 31.958 -8.955 175 ILE CB -2.953 30.524 -11.419 175 ILE CG1 -3.857 29.978 -12.524 175 ILE CG2 -1.451 30.089 -11.512 175 ILE CD1 -3.692 30.529 -13.946 176 ALA N -2.220 30.028 -7.928 176 ALA CA -1.335 30.517 -6.870 176 ALA C 8.120 30.301 -7.310 176 ALA O 0.453 29.215 -7.838 176 ALA CB -1.639 29.838 -5.841 177 VAL N 0.864 31.410 -7.180 177 VAL CA 2.261 31.534 -7.856 177 VAL C 3.225 31.693 -6.473 177 VAL O 3.178 32.657 -8.721 177 VAL CB 2.439 32.607 -8.755 177 VAL CG1 3.842 32.667 -9.592 177 VAL CG2 1.374 32.552 -9.845 178 GLY N 4.877 30.654 -6.398 178 GLY CA 5.168 30.703 -5.339 178 GLY C 6.446 31.233 -6.398 178 GLY O 6.499 31.435 -7.286 179 ALA N 7.812 31.447 -3.287 179 ALA CA 8.713 32.037 -3.859 179 ALA C 9.939 31.099 -5.779 179 ALA C 10.198 30.481 -4.719 179 ALA CB 9.025 33.251 -4.973 180 VAL N 10.659 31.162 -6.885 180 VAL CA 11.970 30.482 -6.981 180 VAL C 13.048 31.585 -7.171 180 VAL O 12.712 32.691 -7.627 180 VAL CB 12.075 29.514 -8.166 180 VAL CG1 11.271 28.251 -7.855 180 VAL CG2 11.675 30.129 -9.800 181 ASP N 14.267 31.203 -6.800 181 ASP CA 13.451 32.108 -7.039 181 ASP C 15.942 31.804 -8.462 181 ASP O 13.339 31.890 -9.292 181 ASP CE 16.446 31.921 -5.914 181 ASP CG 17.128 30.534 -5.971 181 ASP OD1 17.105 29.785 -6.972 181 ASP OD2 17.680 30.256 -4.587 182 SER N 17.087 32.386 -8.847 182 SER CA 17.622 32.214 -10.291 182 SER C 18.153 30.817 -18.494 182 SER O 18.365 30.452 -11.670 182 SER CB 18.678 33.313 -9.423 182 SER OG 18.016 34.561 -10.475 183 SER N 18.258 30.042 -9.423 183 SER CA 18.716 28.645 -9.444 183 SER C 17.881 27.614 -9.547 183 SER O 17.859 26.415 -9.397 183 SER CB 19.256 28.323 -8.007 183 SER OG 28.589 28.615 -8.251 184 ASN N 16.373 28.094 -9.682 184 ASN CA 15.144 27.317 -9.580 184 ASN C 14.931 26.720 -8.197 184 ASN O 14.138 25.759 -8.897 184 ASN CB 15.014 26.341 -10.722 184 ASN CG 14.980 24.090 -12.076 184 ASN OD1 14.700 28.184 -12.277 184 ASN ND2 15.352 24.230 -13.076 185 GLN N 15.542 27.247 -7.159 185 GLN CA 15.276 26.646 -5.835 185 GLN C 14.200 27.494 -5.203 185 GLN O 14.189 28.726 -5.386 185 GLN CB 16.599 26.568 -5.101 185 GLN CG 16.539 26.242 -3.614 185 GLN CD 18.011 26.102 -3.206 185 GLN OE1 18.864 25.798 -4.861 185 GLN NE2 18.266 26.386 -1.934 186 ARG N 13.278 26.958 -4.448 186 ARG CA 12.185 27.774 -1.842 186 ARG C 12.780 28.782 -2.866 186 ARG O 13.698 28.384 -2.893 186 ARG CB 11.213 26.843 -3.116 186 ARG CG 10.214 27.471 -2.161 186 ARG CD 9.467 26.337 -1.469 186 ARG NE 9.866 26.333 -0.117 186 ARG CZ 9.961 26.879 1.039 186 ARG NH1 9.367 27.880 1.658 186 ARG NH2 10.966 26.321 1.783 187 ALA N 12.294 30.009 -2.853 187 ALA CA 12.728 31.064 -1.895 187 ALA C 12.262 30.604 -8.517 187 ALA O 11.196 30.043 -0.387 187 ALA CE 12.144 32.402 -2.344 188 SER N 13.091 30.770 8.549 188 SER CA 12.671 30.286 1.868 188 SER C 11.356 30.847 2.412 188 SER O 10.740 30.111 3.212 188 SER CB 13.767 30.456 2.938 188 SER OG 14.137 31.826 2.841 189 PHE N 10.943 32.010 1.976 189 PHE CA 9.697 32.688 2.418 189 PHE C 8.499 32.198 1.609 189 PHE O 7.389 32.556 2.011 189 PHE CB 9.787 34.217 2.243 189 PHE CG 10.117 34.696 8.867 189 PHE CD1 9.147 34.830 -9.121 189 PHE CD2 11.418 34.116 0.567 189 PHE CE1 9.483 35.187 -1.411 189 PHE CE2 11.769 35.545 -8.701 189 PHE CZ 10.786 35.586 -1.728 190 SER N 8.703 31.526 8.499 190 SER CA 7.626 31.096 -0.391 190 SER C 6.863 30.162 8.328 190 SER O 7.034 29.083 8.866 190 SER CB 8.181 30.590 -1.788 190 SER OG 7.134 30.337 -2.618 191 SER N 5.588 30.551 0.326 191 SER CA 4.341 29.696 0.987 191 SER C 4.261 28.330 0.223 191 SER O 4.543 28.268 -0.995 191 SER CB 3.015 30.411 0.911 191 SER OG 2.729 31.285 1.954 192 VAL N 3.754 27.310 0.928 192 VAL CA 3.629 25.932 0.391 192 VAL C 2.254 25.291 0.686 192 VAL O 1.559 25.698 1.598 192 VAL CB 4.781 25.127 1.888 192 VAL CG1 6.144 25.727 0.722 192 VAL CG2 4.617 25.104 2.592 193 GLY N 1.938 24.172 0.047 193 GLY CA 0.629 23.564 0.410 193 GLY C 0.081 23.029 -0.901 193 GLY O 0.530 23.244 -2.815 194 PRO N -1.023 22.289 -0.722 194 PRO CA -1.662 21.651 -1.873 194 PRO C -2.237 22.605 -2.914 194 PRO O -2.403 22.244 -4.085 194 PRO CB -2.769 20.783 -1.218 194 PRO CG -2.311 20.622 0.213 194 PRO CD -1.633 21.954 0.578 195 GLU N -2.522 23.793 -4.858 195 GLU CA -3.145 24.850 -3.252 195 GLU C -2.095 23.631 -4.858 195 GLU O -2.516 26.398 -4.936 195 GLU CB -4.043 25.786 -2.479 195 GLU CG -4.942 25.134 -1.435 195 GLU CD -4.313 24.860 -0.100 195 GLU DE1 -3.110 24.960 0.165 195 GLU DE2 -3.138 24.520 0.783 196 LEU N -0.129 23.264 -3.870 196 LEU CA 0.241 25.929 -4.666 196 LEU C 0.228 25.376 -6.059 196 LEU O 9.305 24.121 -6.183 196 LEU CB 1.540 25.739 -3.854 196 LEU CG 2.770 26.178 -4.643 196 LEU CD1 2.739 27.716 -4.439 196 LEU CD2 4.027 25.721 -3.911 197 ASP N 0.140 26.208 -7.093 197 ASP CA 0.032 25.774 -8.480 197 ASP C 1.307 25.738 -9.293 197 ASP O 1.655 24.734 -8.914 197 ASP CB -1.067 26.598 -9.191 197 ASP CG -2.406 26.351 -8.549 197 ASP OD1 -2.804 23.158 -8.354 197 ASP OD2 -3.035 27.327 -8.088 198 VAL N 2.013 26.889 -9.344 198 VAL CA 3.206 26.970 -18.209 198 VAL C 4.157 27.950 -9.514 198 VAL O 3.752 28.699 -8.587 198 VAL CB 2.634 27.476 -11.637 198 VAL CG1 1.930 26.724 -12.537 198 VAL CG2 2.337 28.919 -11.484 199 MET N 8.374 27.916 -18.816 199 MET CA 6.430 28.802 -9.498 199 MET C 6.848 29.810 -18.578 199 MET O 6.696 29.518 -11.793 199 MET CB 7.660 27.970 -9.877 199 MET CG 7.865 26.849 -8.139 199 MET SO 6.788 27.469 -6.568 199 MET CB 8.227 27.755 -5.587 200 ALA N 7.426 30.942 -18.103 200 ALA CA 7.991 31.929 -11.055 200 ALA C 9.088 32.666 -10.272 200 ALA O 9.127 32.824 -9.060 200 ALA CB 6.932 32.879 -11.638 201 PRO N 9.927 38.459 -10.981 201 PRO CA 11.813 34.130 -10.238 201 PRO C 10.450 35.127 -9.230 201 PRO O 9.579 35.907 -9.681 201 PRO CB 11.027 34.723 -11.400 201 PRO CG 11.392 34.048 -12.678 201 PRO CD 9.941 31.616 -12.403 202 GLY N 10.925 31.284 -8.021 202 GLY CA 10.473 36.204 -7.044 202 GLY C 11.580 36.698 -6.118 202 GLY O 11.352 37.124 -4.979 203 VAL N 12.019 36.503 -6.618 203 VAL CA 13.948 36.929 -8.716 203 VAL C 14.786 38.017 -6.469 203 VAL O 15.133 37.731 -7.593 203 VAL CB 14.814 35.688 -8.881 203 VAL CG1 16.896 36.106 -4.612 203 VAL CG2 14.879 34.741 -4.878 204 SER N 14.865 39.182 -5.839 204 SER CA 15.872 40.281 -6.487 204 SER C 15.067 40.619 -7.872 204 SER O 15.786 40.685 -8.889 204 SER CB 17.987 39.976 -6.326 204 SER OG 17.732 41.186 -6.672 205 ILE N 13.771 40.865 -8.008 205 ILE CA 13.869 41.234 -9.228 205 ILE C 13.207 42.749 -9.478 205 ILE O 12.675 43.498 -8.648 205 ILE CB 11.832 40.833 -9.144 205 ILE CG1 11.436 39.336 -8.810 205 ILE CG2 10.899 41.281 -10.467 205 ILE CD1 12.257 38.412 -9.771 206 GLN N 13.956 43.095 -10.489 206 GLN CA 16.204 44.517 -10.834 206 GLN C 13.002 44.978 -11.630 206 GLN O 12.669 44.318 -12.621 206 GLN CB 13.455 44.709 -11.740 206 GLN CO 16.684 44.163 -10.980 206 GLN CD 17.285 43.145 -10.007 206 GLN DE1 18.328 44.936 -9.353 206 GLN NE2 16.556 46.260 -9.857 207 SER N 12.359 46.064 -11.214 207 SER CA 11.217 46.571 -11.987 207 SER C 11.089 48.093 -11.749 207 SER O 11.919 48.657 -11.004 207 SER CB 9.918 48.853 -11.869 207 SER OG 8.993 46.056 -12.613 208 THR N 10.054 48.684 -12.526 208 THR CG2 9.171 50.839 -14.754 208 THR OG1 7.870 49.414 -13.144 208 THR CB 8.620 50.415 -13.357 208 THR CA 9.675 50.092 -12.171 208 THR C 9.197 50.488 -10.803 208 THR O 8.423 49.807 -10.849 209 LEU N 9.656 51.618 -10.228 209 LEU CA 9.192 52.158 -8.959 209 LEU C 8.671 53.610 -9.262 209 LEU O 9.140 84.227 -10.222 209 LEU CB 10.335 52.192 -7.958 209 LEU CG 10.804 80.816 -7.416 209 LEU CD1 11.968 51.114 -6.472 209 LEU CD2 9.607 80.282 -6.649 210 PRO N 7.790 54.139 -8.444 210 PRO CA 7.273 55.517 -8.649 210 PRO C 8.383 56.573 -8.639 210 PRO O 9.491 86.445 -8.104 210 PRO CB 6.302 55.733 -7.517 210 PRO CG 6.004 54.379 -6.944 210 PRO CD 7.193 53.491 -7.271 211 GLY N 8.077 87.665 -9.355 211 GLY CA 9.069 58.763 -9.410 211 GLY C 10.094 88.454 -18.490 211 GLY O 11.176 59.005 -10.259 212 ASN N 9.851 87.770 -11.887 212 ASN CA 10.903 57.422 -12.643 212 ASN C 12.059 86.733 -12.856 212 ASN O 13.188 57.181 -12.420 212 ASN CB 11.224 88.395 -13.499 212 ASN CG 11.803 58.185 -14.814 212 ASN OD1 11.853 87.854 -13.323 212 ASN ND2 12.273 59.159 -15.576 213 LYS N 11.803 89.749 -11.247 213 LYS CA 12.610 54.946 -10.337 213 LYS C 12.668 83.459 -10.866 213 LYS O 11.773 53.039 -11.613 213 LYS CB 12.769 88.241 -9.059 213 LYS CG 13.206 56.694 -8.767 213 LYS CD 13.246 87.030 -7.312 214 TYR C 14.383 50.600 -9.489 214 TYR O 15.211 81.253 -8.817 214 TYR CB 14.641 80.981 -11.984 214 TYR CG 14.130 81.621 -13.246 214 TYR CD1 14.689 82.847 -13.678 214 TYR CD2 18.129 81.063 -14.814 214 TYR CE1 14.230 83.475 -14.814 214 TYR CE2 12.654 81.669 -15.178 214 TYR CZ 13.204 82.895 -15.550 214 TYR DM 12.756 83.458 -16.696 215 GLY N 14.058 48.847 -9.158 215 GLY CA 14.622 48.772 -7.903 215 GLY C 14.138 49.325 -7.749 215 GLY B 18.849 46.917 -8.321 216 ALA N 14.810 46.638 -6.831 216 ALA CA 14.454 45.203 -6.781 216 ALA C 13.682 44.922 -5.512 216 ALA O 13.948 45.527 -4.475 216 ALA CB 15.713 44.854 -6.887 217 TYR N 12.758 43.982 -5.575 217 TYR CA 11.964 43.488 -4.440 217 TYR C 12.033 41.928 -4.547 217 TYR O 12.202 41.442 -5.656 217 TYR CE 10.473 43.862 -4.578 217 TYR CG 10.117 45.291 -4.214 217 TYR CD1 18.846 45.981 -3.236 217 TYR CD2 9.016 45.933 -4.785 217 TYR CE1 18.459 47.267 -2.790 217 TYR CE2 8.654 47.210 -4.381 217 TYR CZ 9.358 47.882 -3.391 217 TYR OH 8.953 48.160 -2.988 218 ASN N 11.758 41.386 -3.891 218 ASN CA 11.640 39.941 -3.227 218 ASN C 18.804 89.636 -2.748 218 ASN O 9.763 40.347 -1.917 218 ASN CB 12.953 39.348 -2.154 218 ASN CG 14.831 39.566 -2.843 218 ASN DD1 14.612 39.709 -3.342 218 ASN ND2 14.660 39.644 -1.168 219 GLY N 9.678 38.854 -3.289 219 GLY CA 8.382 30.130 -2.649 219 GLY C 7.870 37.384 -3.681 219 GLY O 7.873 37.502 -4.876 220 THR N 6.861 86.430 -3.283 220 THR CA 8.697 35.036 -4.179 220 THR C 6.879 87.044 -4.864 220 THR O 6.417 36.742 -5.958 220 THR CB 6.825 84.819 -3.526 220 THR OG1 6.136 38.543 -2.451 220 THR CG2 3.784 83.696 -2.980 221 SER N 4.738 38.238 -4.303 221 SER CA 3.984 89.201 -5.149 221 SER C 4.768 39.641 -6.383 221 SER O 4.117 40.208 -7.277 221 SER CB 8.323 40.383 -4.546 221 SER OG 3.435 48.282 -3.149 222 MET N 6.060 39.389 -6.485 222 MET CE 6.471 42.771 -5.193 222 MET SD 7.768 41.333 -4.993 222 MET CG 8.506 41.399 -6.602 222 MET CB 8.351 40.015 -7.218 222 MET CA 6.916 39.670 -7.638 222 MET C 6.877 38.435 -8.567 222 MET O 7.084 38.567 -9.775 223 ALA N 6.554 37.246 -8.041 223 ALA CA 6.469 36.028 -8.885 223 ALA C 8.200 86.068 -9.707 223 ALA O 5.133 35.948 -10.929 223 ALA CB 6.509 84.807 -7.923 224 SER N 4.076 36.360 -9.038 224 SER CA 2.738 86.488 -9.700 224 SER C 2.661 37.161 -11.039 224 SER O 2.145 36.393 -12.057 224 SER CB 1.801 36.995 -8.603 224 SER OG 8.492 36.099 -9.157 225 PRO N 3.256 38.411 -11.139 225 PRO CA 8.895 39.130 -12.439 225 PRO C 3.764 38.469 -13.626 225 PRO O 8.406 38.650 -14.804 225 PRO CB 3.653 40.511 -12.954 225 PRO CG 4.411 40.402 -10.764 225 PRO CD 3.735 39.224 -10.054 226 HIS N 4.769 37.626 -13.299 226 HIS CA 5.446 36.879 -14.362 226 HIS C 4.618 35.947 -15.061 226 HIS O 4.425 35.809 -16.293 226 HIS CB 6.608 36.046 -13.765 226 HIS CG 7.814 36.059 -13.358 226 HIS ND1 6.848 37.488 -12.170 226 HIS CD2 8.883 37.118 -14.167 226 HIS CE1 9.270 38.052 -12.236 226 HIS NE2 9.771 37.866 -13.463 227 VAL N 3.593 35.366 -14.199 227 VAL CA 2.583 34.388 -14.727 227 VAL C 1.479 35.197 -15.421 227 VAL O 1.018 34.773 -16.490 227 VAL CB 2.203 33.444 -13.619 227 VAL CG1 1.076 32.476 -14.246 227 VAL CG2 3.204 32.665 -12.891 228 ALA N 1.003 36.242 -14.814 228 ALA CA 8.011 37.109 -15.517 228 ALA C 0.843 37.538 -16.868 228 ALA O -0.253 37.435 -17.828 228 ALA CB -0.307 38.353 -14.668 229 GLY N 1.791 38.028 -16.941 229 GLY CA 2.352 38.408 -18.239 229 GLY C 2.420 37.197 -19.187 229 GLY O 2.189 37.375 -20.384 230 ALA N 2.711 35.988 -18.646 230 ALA CA 2.794 34.801 -18.546 230 ALA C 1.424 34.500 -20.153 230 ALA O 1.380 34.205 -21.343 230 ALA CB 3.298 33.624 -18.709 231 ALA N 0.385 34.623 -19.328 231 ALA CA -1.010 34.416 -19.744 231 ALA C -1.256 35.423 -20.064 231 ALA O -1.909 33.056 -21.852 231 ALA CB -1.932 34.664 -18.849 232 ALA N -0.778 36.657 -20.721 232 ALA CA -1.013 37.663 -21.792 232 ALA C -0.201 37.284 -23.078 232 ALA O -0.841 37.501 -24.187 232 ALA CB -8.742 39.121 -21.377 233 LEU N 0.935 36.724 -22.967 233 LEU CA 1.617 36.293 -24.209 233 LEU C 0.821 35.169 -24.880 233 LEU O 0.696 35.231 -26.111 233 LEU CB 3.063 35.877 -23.907 233 LEU CG 3.996 36.994 -23.453 233 LEU CD1 5.259 36.342 -22.921 233 LEU CD2 4.241 37.053 -24.680 234 ILE N 0.357 34.199 -24.047 234 ILE CD1 0.306 30.664 -21.657 234 ILE CG1 8.454 31.223 -23.105 234 ILE CB -0.811 32.014 -23.570 234 ILE CG2 -1.803 30.900 -24.091 234 ILE CA -0.406 33.076 -24.644 234 ILE C -1.621 33.997 -25.434 234 ILE O -1.883 33.144 -26.544 235 LEU N -2.390 34.465 -24.779 235 LEU CA -3.596 35.028 -25.423 235 LEU C -3.258 35.843 -26.672 235 LEU O -4.109 35.914 -27.589 235 LEU CE -4.432 35.765 -24.378 235 LEU CG -5.140 34.899 -23.342 235 LEU CD1 -5.652 35.683 -22.145 235 LEU CD2 -6.252 34.138 -24.120 236 SER N -2.094 36.438 -26.798 236 SER CA -1.764 37.237 -27.986 236 SER C -1.491 36.292 -29.144 236 SER O -1.746 36.634 -30.290 236 SER CB -0.433 38.234 -27.733 236 SER OG 0.599 37.571 -27.582 237 LYS N -1.046 35.067 -28.882 237 LYS CA -0.846 34.055 -29.952 237 LYS C -2.113 33.277 -30.269 237 LYS O -2.378 32.951 -31.664 237 LYS CB 0.272 33.112 -29.551 237 LYS CG 0.677 32.240 -30.716 237 LYS CD 2.928 31.535 -30.442 237 LYS CE 2.345 30.762 -31.724 237 LYS NZ 3.528 29.048 -31.596 238 HIS N -2.951 31.989 -29.310 238 HIS CA -4.168 32.163 -29.379 238 HIS C -6.334 32.899 -28.697 238 HIS O -5.713 32.584 -27.562 238 HIS CB -3.948 30.062 -28.311 238 HIS CG -3.809 29.921 -29.237 238 HIS ND1 -1.707 29.679 -28.835 238 HIS CD2 -3.137 29.258 -30.394 238 HIS CE1 -1.084 28.851 -29.642 238 HIS NE2 -1.948 28.600 -30.599 239 PRO N -5.048 33.917 -29.365 239 PRO CA -6.900 34.778 -28.773 239 PRO C -8.204 34.052 -28.532 239 PRO O -8.949 34.519 -27.662 239 PRO CB -7.018 35.977 -29.713 239 PRO CG -6.666 35.294 -31.827 239 PRO CD -5.436 34.434 -30.468 240 ASN N -8.306 32.969 -29.227 240 ASN CA -9.529 32.041 -29.216 240 ASN C -9.508 31.180 -27.980 240 ASN O -10.340 30.610 -29.216 240 ASN CB -9.403 31.249 -30.535 240 ASN CG -7.971 30.827 -30.889 240 ASN OD1 -7.008 31.590 -31.147 240 ASN ND2 -7.670 29.509 -30.926 241 TRP N -8.354 31.806 -27.304 241 TRP CA -8.304 30.124 -26.120 241 TRP C -9.106 30.638 -24.936 241 TRP O -9.843 31.833 -24.686 241 TRP CB -6.879 29.830 -25.679 241 TRP CG -6.094 28.903 -26.557 241 TRP CD1 -6.338 28.433 -27.818 241 TRP CD2 -6.039 28.324 -26.155 241 TRP NE1 -5.362 27.547 -28.211 241 TRP CE2 -4.414 27.676 -27.216 241 TRP CE3 -4.097 28.406 -26.981 241 TRP CZ2 -3.195 26.786 -27.174 241 TRP CZ3 -2.912 27.667 -26.943 241 TRP CH2 -2.470 26.873 -26.005 242 THR N -9.727 29.781 -24.142 242 THR CA -10.438 30.119 -22.911 242 THR C -9.669 30.176 -21.747 242 THR O -8.335 29.676 -21.937 242 THR CB -11.579 29.032 -22.675 242 THR OG1 -10.837 27.786 -22.476 242 THR CG2 -12.494 28.907 -23.895 243 ASN N -9.946 30.659 -20.611 243 ASN ND2 -11.787 30.404 -18.747 243 ASN OD1 -11.465 31.518 -16.788 243 ASN CG -11.093 31.131 -17.905 243 ASN CB -9.708 31.530 -18.332 243 ASN CA -9.053 30.731 -19.444 243 ASN C -8.657 29.303 -19.010 243 ASN O -7.593 29.136 -18.440 244 THR N -9.964 28.362 -19.283 244 THR CA -9.381 26.934 -19.059 244 THR C -8.133 26.393 -19.802 244 THR O -7.324 25.757 -19.111 244 THR CB -10.865 26.088 -19.494 244 THR OG1 -11.735 26.675 -18.684 244 THR CG2 -10.503 24.595 -19.158 245 GLN N -8.082 26.716 -21.073 245 GLN CA -6.964 26.362 -21.962 245 GLN C -5.647 27.020 -21.520 245 GLN O -4.573 26.393 -21.447 245 GLN CB -7.330 26.599 -23.397 245 GLN CG -8.265 25.526 -23.989 245 GLN CD -8.493 25.873 -23.428 245 GLN DE1 -9.306 26.769 -25.727 245 GLN NE2 -7.745 25.312 -26.370 246 VAL N -5.697 28.304 -21.218 246 VAL CA -6.677 29.048 -20.778 246 VAL C -3.936 26.462 -19.467 246 VAL O -2.705 28.227 -19.361 246 VAL CB -4.779 30.555 -20.621 246 VAL CG1 -3.544 31.272 -20.027 246 VAL CG2 -5.169 31.138 -21.959 247 ARG N -4.767 28.240 -18.462 247 ARG CA -4.380 27.714 -17.168 247 ARG C -3.770 26.252 -17.360 247 ARG O -2.705 25.985 -16.764 247 ARG CB -5.833 27.667 -16.149 247 ARG CG -4.987 27.095 -14.852 247 ARG CD -6.056 27.179 -13.793 247 ARG NE -5.440 26.757 -12.546 247 ARG CZ -5.893 26.866 -11.313 247 ARG NM1 -7.064 27.484 -11.210 247 ARG NM2 -3.177 26.428 -10.270 248 SER N -4.480 25.505 -18.131 248 SER CA -4.039 24.131 -18.426 248 SER C -2.657 24.086 -19.073 248 SER O -1.848 23.253 -18.583 248 SER CB -5.034 23.408 -19.372 248 SER OG -6.146 23.090 -18.832 249 SER N -2.500 24.853 -20.136 249 SER CA -1.223 24.874 -20.851 249 SER C -0.071 23.302 -19.940 249 SER O 2.026 24.705 -20.049 249 SER CB -1.369 25.758 -22.068 249 SER OG -0.380 25.419 -22.956 250 LEU N -8.289 26.833 -19.160 250 LEU CD2 1.824 29.814 -18.222 250 LEU CD1 -0.373 30.453 -17.268 250 LEU CG 0.352 29.438 -18.151 250 LEU CB 0.178 28.063 -17.503 250 LEU CA 0.718 26.837 -18.216 250 LEU C 1.092 25.694 -17.265 250 LEU O 2.283 25.421 -17.032 251 GLN N 8.068 25.007 -16.914 251 GLN NE2 -2.750 25.512 -12.237 251 GLN DE1 -2.819 23.424 -12.935 251 GLN CD -2.345 24.510 -13.034 251 GLN CG -1.218 24.814 -13.994 251 GLN CB -0.857 23.621 -14.877 251 GLN CA 0.381 23.941 -15.745 251 GLN C 0.959 22.664 -16.361 251 GLN O 1.743 22.014 -15.616 252 ASN N 0.633 22.394 -17.390 252 ASN CA 1.082 21.204 -18.282 252 ASN C 2.394 21.359 -18.991 252 ASN B 2.809 20.442 -18.768 252 ASN CB 0.004 20.780 -19.292 252 ASN CG -1.036 19.926 -18.573 252 ASN OD1 -0.036 19.355 -17.582 252 ASN ND2 -2.234 19.834 -19.161 253 THR N 3.018 22.505 -18.923 253 THR CA 4.256 22.717 -19.713 253 THR C 9.381 23.247 -18.816 253 THR O 6.348 25.733 -19.427 253 THR CB 4.086 23.672 -20.952 253 THR OG1 3.395 24.957 -20.428 253 THR CG2 3.147 23.138 -22.032 254 THR N 3.218 23.177 -17.351 254 THR CA 6.216 23.612 -16.588 254 THR C 7.466 22.700 -16.612 254 THR O 7.402 21.580 -17.095 254 THR CB 5.664 23.558 -13.132 254 THR OG1 5.129 22.178 -15.046 254 THR CG2 4.530 24.549 -14.802 255 THR N 8.499 23.296 -16.876 255 THR CA 9.771 22.594 -15.817 255 THR C 9.621 22.031 -14.414 255 THR O 9.439 22.786 -13.474 255 THR CB 11.080 23.455 -15.897 255 THR OG1 11.082 23.709 -17.321 255 THR CG2 12.286 22.628 -15.486 256 LYS N 9.606 20.702 -14.314 256 LYS CA 9.364 20.063 -13.818 256 LYS C 30.522 20.333 -12.063 256 LYS O 11.662 20.274 -12.592 256 LYS CB 9.024 18.598 -13.249 256 LYS CB 9.018 17.805 -11.921 256 LYS CD 10.286 16.948 -11.777 256 LYS CE 10.212 15.940 -10.623 256 LYS NZ 9.243 16.869 -11.054 257 LEU N 10.212 20.674 -10.824 257 LEU CA 11.272 21.036 -9.893 257 LEU C 11.250 20.232 -8.614 257 LEU O 12.096 20.565 -7.732 257 LEU CB 11.187 22.547 -9.522 257 LEU CG 11.357 23.620 -10.568 257 LEU CD1 11.245 25.803 -9.921 257 LEU CD2 12.678 23.468 -11.325 258 GLY N 10.431 19.282 -8.298 258 GLY CA 10.602 15.793 -6.879 258 GLY C 9.168 18.703 -6.373 258 GLY O 8.283 18.956 -7.202 259 ASP N 9.024 18.202 -5.150 259 ASP CA 7.757 17.896 -4.516 259 ASP C 6.659 18.941 -4.789 259 ASP O 6.859 20.039 -4.214 259 ASP CB 7.996 17.840 -3.053 259 ASP CG 6.781 17.128 -2.241 259 ASP OD1 5.611 17.527 -2.354 259 ASP OD2 7.098 16.299 -1.321 260 SER N 5.560 18.610 -5.312 260 SER CA 4.481 19.587 -5.529 260 SER C 4.046 20.362 -4.289 260 SER O 3.500 21.503 -4.446 260 SER CB 3.345 18.919 -6.289 260 SER OG 2.745 17.937 -5.448 261 PHE N 4.241 19.778 -3.112 261 PHE CA 3.831 20.468 -1.885 261 PHE C 6.544 21.846 -1.863 261 PHE O 3.944 22.848 -1.432 261 PHE CB 4.053 19.749 -0.563 261 PHE CG 3.549 20.337 0.715 261 PHE CD1 2.206 20.163 1.125 261 PHE CD2 4.401 21.060 1.559 261 PHE CE1 1.737 20.717 2.315 261 PHE CE2 3.945 21.602 2.748 261 PHE CZ 2.605 21.465 3.114 262 TYR N 5.778 21.758 -2.305 262 TYR CA 6.688 22.914 -2.251 262 TYR C 6.820 23.689 -3.545 262 TYR O 7.201 24.853 -3.393 262 TYR CB 8.123 22.455 -1.851 262 TYR CG 8.146 21.092 -0.454 262 TYR CD1 8.034 20.484 -0.364 262 TYR CD2 8.149 22.669 0.698 262 TYR CE1 8.062 19.873 0.882 262 TYR CE2 8.114 22.069 1.962 262 TYR CZ 8.069 20.672 2.018 262 TYR OH 7.963 20.029 3.205 263 TYR N 6.626 23.104 -4.693 263 TYR CA 6.812 23.655 -6.022 263 TYR C 5.626 23.680 -6.956 263 TYR O 5.781 24.117 -8.111 263 TYR CB 7.928 22.768 -6.681 263 TYR CG 9.279 23.035 -6.068 263 TYR CD1 10.064 24.046 -6.637 263 TYR CD2 9.800 22.342 -4.995 263 TYR CE1 11.335 24.328 -6.168 263 TYR CE2 11.062 22.640 -6.491 263 TYR CZ 11.838 23.618 -5.106 263 TYR OH 13.063 23.949 -6.597 264 GLY N 4.471 23.161 -6.516 264 GLY CB 3.301 23.064 -7.412 264 GLY C 3.847 22.196 -8.556 264 GLY O 4.647 21.274 -8.365 265 LYS N 3.436 22.477 -9.754 265 LYS CA 3.834 21.798 -10.971 265 LYS C 5.188 22.232 -11.464 265 LYS O 5.684 21.563 -12.386 265 LYS CB 2.733 22.071 -12.844 265 LYS CG 1.490 21.563 -11.305 265 LYS CD 0.710 20.548 -12.079 265 LYS CE -0.692 20.496 -11.391 265 LYS NZ -1.678 20.757 -12.489 266 GLY N 5.787 23.226 -10.817 266 GLY CA 7.120 23.612 -11.325 266 GLY C 7.155 25.052 -11.818 266 GLY O 6.177 25.793 -11.648 267 LEU N 8.262 25.336 -12.480 267 LEU CA 8.490 26.640 -13.097 267 LEU C 7.804 26.771 -14.437 267 LEU O 7.953 25.909 -15.298 267 LEU CB 10.910 26.855 -13.214 267 LEU CG 10.432 28.060 -14.098 267 LEU CD1 10.096 29.331 -13.250 267 LEU CD2 11.924 27.921 -14.327 268 ILE N 7.064 27.863 -14.832 268 ILE CA 6.406 28.035 -13.944 268 ILE C 7.426 28.246 -17.065 268 ILE O 8.539 28.793 -16.912 268 ILE CB 5.369 29.218 -15.899 268 ILE CG1 6.099 30.541 -15.592 268 ILE CG2 4.243 26.925 -14.567 268 ILE CD1 5.399 31.765 -16.262 269 ASN N 7.897 27.843 -18.237 269 ASN CA 7.802 27.975 -19.437 269 ASN C 6.839 20.584 -28.495 269 ASN O 8.945 27.760 -20.943 269 ASN CB 8.432 26.653 -19.895 269 ASN CG 9.161 24.806 -21.210 269 ASN OD1 8.991 27.626 -22.122 269 ASN ND2 10.011 25.796 -21.472 270 VAL N 6.908 29.868 -20.724 270 VAL CA 5.863 30.418 -21.614 270 VAL C 6.039 30.907 -23.054 270 VAL O 5.097 27.969 -23.972 270 VAL CB 5.656 31.950 -21.422 270 VAL CG1 6.849 32.797 -21.874 270 VAL CG2 4.428 32.362 -22.832 271 GLN N 7.325 29.701 -23.352 271 GLN CA 7.603 29.278 -24.744 271 GLN C 6.869 27.934 -25.031 271 GLN O 6.213 27.806 -26.091 271 GLN CB 9.104 25.220 -24.964 271 GLN CG 9.486 28.618 -26.338 271 GLN CD 10.901 28.585 -26.582 271 GLN DE1 11.369 28.379 -27.718 271 GLN NE2 11.702 28.553 -25.510 272 ALA N 6.977 26.999 -24.892 272 ALA CA 6.224 25.712 -24.240 272 ALA C 4.701 25.950 -24.164 272 ALA O 3.898 25.505 -25.001 272 ALA CB 6.743 24.742 -23.172 273 ALA N 4.247 26.661 -23.135 273 ALA CA 2.840 26.921 -22.859 273 ALA C 2.081 27.528 -24.020 273 ALA O 0.899 27.219 -24.255 273 ALA CB 2.736 27.773 -21.585 274 ALA N 2.755 28.484 -24.762 274 ALA CB 2.952 30.391 -26.210 274 ALA CA 2.109 29.144 -25.847 274 ALA C 1.730 28.367 -27.090 274 ALA O 0.980 28.949 -27.921 275 GLN N 2.350 27.194 -27.314 275 GLN CA 2.048 26.389 -28.527 275 GLN C 2.147 27.261 -29.777 275 GLN O 3.260 27.807 -29.916 275 GLN OT 1.193 27.361 -30.590 275 GLN CB 0.666 25.734 -28.520 275 GLN CG 0.501 24.664 -27.447 275 GLN CD -0.823 23.934 -27.631 275 GLN DE1 -1.376 23.895 -28.729 275 GLN NE2 -1.373 23.411 -26.538

The above structural studies together with the above referenced kinetic data and kinetic data presented herein indicate that the subsites in the binding cleft of subtilisin are capable of interacting with substrate amino acid residues from P-4 to P-2′.

The most extensively studied of the above residues are Gly166, Gly169 and Ala152. There amino acids were identified as residues within the S-1 subsite. As seen in FIG. 3, which is a stereoview of the S-1 subsite, Gly166 and Gly169 occupy positions at the bottom of the S-1 subsite, whereas Ala152 occupies a position near the top of S-1, close to the catalytic Ser221.

All 19 amino acid substitutions of Gly166 and Gly169 have been made. As will be indicated in the examples which follow, the preferred replacement amino acids for Gly166 and/or Gly169 will depend on the specific amino acid occupying the P-1 position of a given substrate.

The only substitutions of Ala152 presently made and analyzed comprise the replacement of Ala152 with Gly and Ser. The results of these substitutions on P-1 specificity will be presented in the examples.

In addition to those residues specifically associated with specificity for the P-1 substrate amino acid, Tyr104 has been identified as being involved with P-4 specificity. Substitutions at Phe189 and Tyr217, however, are expected to respectively effect P-2′ and P-1′ specificity.

The catalytic activity of subtilisin has also been modified by single amino acid substitutions at Asn155.

The catalytic triad of subtilisin is shown in FIG. 4. As can be seen, Ser221, His64 and Asp32 are positioned to facilitate nucleophilic attach by the serine hydoxylate on the carbonyl of the scissile peptide bond. Several hydrogen bonds may also help to stabilize the transition state complex for the tetrahedral substrate intermediate. One hydrogen bond is between aspartate and the positively charged histidine, ND1. Kossiakoff, A. A., et al. (1981) Biochem. 20, 6462-5474. A second hydrogen bond forms between the scissile amide nitrogen of the substrate and the (NE2) proton on the histidine. A third set of hydrogen bonds forms between the enzyme and the oxyanion that is produced from the carbonyl oxygen of the substrate. This latter set of hydrogen bonds is formed differently by the mammalian serine proteases and substilisin. A fourth hydrogen bond appears to exist between the amide nitrogen of the peptide bond between P-1 and P-2 and the carbonyl oxygen of Ser125. Specifically, x-ray crystallographic studies of chymotrypsin (Henderson, R. (1970) J. Mol. Biol. 54, 341) indicate that two hydrogen bonds form between the substrate oxyanion and two main-chain amide protons from the enzyme (Gly193 and the catalytic Ser195). Crystallographic studies of subtilisin (Robertus, et al. (1972) Biochem. 11, 4293-4303; Matthews, et al. (1975) J. Biol. Chem. 250, 7120-7126; Poulos, et al. (1976) J. Biol. Chem. 250, 1097-1103) show that two hydrogen bonds are also formed with the oxyanion; one hydrogen bond donor is from the catalytic serine-221 main-chain amide while the other is from one of the NE2 protons of the asparagine-155 side chain. See FIG. 4.

Asn155 was substituted with Ala, Asp, His, Glu and Thr. These substitutions were made to investigate the the stabilization of the charged tetrahedral intermediate of the transition state complex by the potential hydrogen bond between the side chain of Asn155 and the oxyanion of the intermediate. These particular substitutions caused large decreases in substrate turnover, kcat (200 to 4,000 fold), marginal decreases in substrate binding Km (up to 7 fold), and a loss in transition state stabilization energy of 2.2 to 4.7 kcal/mol. The retention of Km and the drop in kcat will make these mutant enzymes useful as binding proteins for specific peptide sequences, the nature of which will be determined by the specificity of the precursor protease.

Various other amino acid residues have been identified which affect alkaline stability. In some cases, mutants having altered alkaline stability also have altered thermal stability.

In B amyloliguefaciens subtilisin residues Asp36, Ile107, Lys170 , Ser204 and Lys213 have been identified as residues which upon substitution with a different amino acid alter the alkaline stability of the mutated enzyme as compared to the precursor enzyme. The substitution of Asp36 with Ala and the substitution of Lys170 with Glu each resulted in a mutant enzyme having a lower alkaline stability as compared to the wild type subtilisin. When Ile107 was substituted with Val, Ser204 substituted with Cys, Arg or Leu or Lys213 substituted with Arg, the mutant subtilisin had a greater alkaline stability as compared to the wild type subtilisin. However, the mutant Ser204P demonstrated a decrease in alkaline stability.

In addition, other residues, previously identified as being associated with the modification of other properties of subtilisin, also affect alkaline stability. These residues include Ser24, Met50, Glu156, Gly166, Gly169 and Tyr217. Specifically the following particular substitutions result in an increased alkaline stability: Ser24C, Met50F, Gly156Q or S, Gly166A, H, K, N or Q, Gly169S or A, and Tyr217 F, K, R or L. The mutant Met50V, on the other hand, results in a decrease in the alkaline stability of the mutant subtilisin as compared to wild type subtilisin.

Other residues involved in alkaline stability based on the alkaline stability screen include the mutants of Table I for residues Asp197 and Met222.

Various other residues have been identified as being involved in thermal stability as determined by the thermal stability screen herein. These residues include the above identified residues which effect alkaline stability and Met199 and Tyr21 . These latter two residues are also believed to be important for alkaline stability. Mutants at these residues include I199 and F21.

The amino acid sequence of B. amyloliguefaciens substilisin has also been modified by substituting two or more amino acids of the wild-type sequence. Six categories of multiply substituted mutant subtilisin have been identified. The first two categories comprise thermally and oxidatively stable mutants. The next three other categories comprise mutants which combine the useful properties of any of several single mutations of B. amyloliguefaciens subtilisin. The last category comprises mutants which have modified alkaline and/or thermal stability.

The first category is double mutants in which two cysteine residues have been substituted at various amino acid residue positions within the subtilisin BPN′ molecule. Formation of disulfide bridges between the two substituted cysteine residues results in mutant subtilisins with altered thermal stability and catalytic activity. These mutants include A21/C22/C87 and C24/C87 which will be described in more detail in Example 11.

The second category of multiple subtilisin mutants comprises mutants which are stable in the presence of various oxidizing agents such as hydrogen peroxide or peracids. Examples 1 and 2 describe these mutants which include F50/I124/Q222, F50/I124, F50/Q222, F50/L124/Q222, I124/Q222 and L124/Q222.

The third category of multiple subtilisin mutants comprises mutants with substitutions at position 222 combined with various substitutions at positions 166 or 169. These mutants, for example, combine the property of oxidative stability of the A222 mutation with the altered substrate specificity of the various 166 or 169 substitutions. Such multiple mutants include A166/A222, A166/C222, F166/C222, K166/A222, K166/C222, V166/A222 and V166/C222. The K166/A222 mutant subtilisin, for example, has a kcat/Km ratio which is approximately two times greater than that of the single A222 mutant subtilisin when compared using a substrate with phenylalanine as the P-1 amino acid. This category of multiple mutant is described in more detail in Example 12.

The fourth category of multiple mutants combines substitutions at position 156 (Glu to Q or S) with the substitution of Lys at position 166. Either of these single mutations improve enzyme performance upon substrates with glutamate as the P-1 amino acid. When these single mutations are combined, the resulting multiple enzyme mutants perform better than either precursor. See Example 9.

The fifth category of multiple mutants contain the substitution of up to four amino acids of the B. amyloliguefaciens subtilisin sequence. These mutants have specific properties which are virtually identicle to the properties of the subtilisin from B. licheniformis. The subtilisin from B. licheniformis differs from B. amyloliguefaciens subtilisin at 87 out of 275 amino acids. The multiple mutant F50/S156/A169/L217 was found to have similar substrate specificity and kinetics to the licheniformis enzyme. (See Example 13.) However, this is probably due to only three of the mutations (S156, A169 and L217) which are present in the substrate binding region of the enzyme. It is quite surprising that, by making only three changes out of the 87 different amino acids between the sequence of the two enzymes, the B. amyloliguifaciens enzyme was converted into an enzyme with properties similar to B. licheniformis enzyme. Other enzymes in this series include F50/Q156/N166/L217 and F50/S156/L217.

The sixth category of multiple mutants includes the combination of substitutions at position 107 (Ile to V) with the substitution of Lys at position 213 with Arg, and the combination of substitutions of position 204 (preferably Ser to C or L but also to all other amino acids) with the substituion of Lys at position 213 with R. Other multiple mutants which have altered alkaline stability include Q156/K166, Q156/N166, S156/K166, S156/N166 (previously identified as having altered substrate specificity), and F50/S156/A169/L217 (previously identified as a mutant of B. amyloliguifaciens subtilisin having properties similar to subtilisin from B. licheniformis). The mutant, F50/V107/R213 was constructed based on the observed increase in alkaline stability for the single mutants F50, V107 and R213. It was determined that the V107/R213 mutant had an increased alkaline stability as compared to the wild type subtilisin. In this particular mutant, the increased alkaline stability was the result of the cumulative stability of each of the individual mutations. Similarly, the mutant F50/V107/R213 had an even greater alkaline stability as compared to the V107/R213 mutant indicating that the increase in the alkaline stability due to the F50 mutation was also cumulative.

Table IV summarizes the multiple mutants which have been made including those not mentioned above.

In addition, based in part on the above results, substitution at the following residues in subtilisin is expected to produce a multiple mutant having increased thermal and alkaline stability: Ser24, Met50, Ile107, Glu156, Gly166, Gly169, Ser204, Lys213, Gly215, and Tyr217.

TABLE IV Triple, Quadruple Double Mutants or Other Multiple C22/C87 F50/1124/Q222 C24/C87 F50/L124/Q222 V45/V48 F50/L124/A222 C49/C94 A21/C22/C87 C49/C95 F50/S156/N166/L217 C50/C95 F50/Q156/N166/L217 C50/C110 F50/S156/A169/L217 F50/1124 F50/S156/L217 F50/Q222 F50/Q156/K166/L217 I124/Q222 F50/S156/K166/L217 Q156/D166 F50/Q156/K166/K217 Q156/K166 F50/S156/K166/K217 Q156/N166 F50/V107/R213 S156/D166 [S153/S156/A158/G159/S160/A161- S156/K166 164/I165/S166/A169/R170] S156/N166 L204/R213 S156/A169 R213/204A, E, Q, D, N, G, K, A166/A222 V, R, T, P, I, M, F, Y, W A166/C222 or H F166/A222 V107/R213 F166/C222 K166/A222 K166/C222 V166/A222 V166/C222 A169/A222 A169/A222 A169/C222 A21/C22

In addition to the above identified amino acid residues, other amino acid residues of subtilisin are also considered to be important with regard to substrate specificity. These are the aforementioned residues which have yet to be mutated. Mutation of each of these residues is expected to produce changes in the substrate specificity of subtilisin. Moreover, multiple mutations among these residues and among the previously identified residues are also expected to produce subtilisin mutants having novel substrate specificity.

Particularly important residues are His67, Ile107, Leu126 and Leu135. Mutation of His67 should alter the S-1′ subsite, thereby altering the specificity of the mutant for the P-1′ substrate residue. Changes at this position could also affect the pH activity profile of the mutant. This residue was identified based on the inventor's substrate modeling from product inhibitor complexes.

Ile107 is involved in P-4 binding. Mutation at this position thus should alter specificity for the P-4 substrate residue. Ile107 was also identified by molecular modeling from product inhibitor complexes.

The S-2 binding site includes the Leu126 residue. Modification at this position should therefore affect P-2 specificity. Moreover, this residue is believed to be important to convert subtilisin to an amino peptidase. The pH activity profile should also be modified by appropriate substitution. These residues were identified from inspection of the refined model, the three dimensional structure from modeling studies. A longer side chain is expected to preclude binding of any side chain at the S-2 subsite. Therefore, binding would be restricted to subsites S-1, S-1′, S-2′, S-3′ and cleavage would be forced to occur after the amino terminal peptide.

Leu135 is in the S-4 subsite and if mutated should alter substrate specificity for P-4 if mutated. This residue was identified by inspection of the three-dimensional structure and modeling based on the product inhibitor complex of F222.

In addition to these sites, specific amino acid residues within the segments 97-103, 126-129 and 213-215 are also believed to be important to substrate binding.

Segments 97-103 and 126-129 form an antiparallel beta sheet with the main chain of substrate residues P-4 through P-2. Mutating residues in those regions should affect the substrate orientation through main chain (enzyme)—main chain (substrate) interactions, since the main chain of these substrate residues do not interact with these particular residues within the S-4 through S-2 subsites.

Within the segment 97-103, Gly97 and Asp99 may be mutated to alter the position of residues 101-103 within the segment. Changes at these sites must be compatible, however. In B. amyloliguifaciens subtilisin Asp99 stabilizes a turn in the main chain tertiary folding that affects the direction of residues 101-103. B. licheniformis subtilisin Asp97, functions in an analogous manner.

In addition to Gly97 and Asp99, Ser101 interacts with Asp99 in B. amyliguefaciens subtilisin to stabilize the same main chain turn. Alterations at this residue should alter the 101-103 main chain direction. Mutations at Glu103 are also expected to affect the 101-103 main chain direction.

The side chain of Gly102 interacts with the substrate P-3 amino acid. Side chains of substituted amino acids thus are expected to significantly affect specificity for the P-3 substrate amino acids.

All the amino acids within the 127-129 segment are considered important to substrate specificity. Gly 127 is positioned such that its side chain interacts with the S-1 and S-3 subsites. Altering this residue thus should alter the specificity for P-1 and P-3 residues of the substrate.

The side chain of Gly128 comprises a part of both the S-2 and S-4 subsites. Altered specificity for P-2 and P-4 therefore would be expected upon mutation. Moreover, such mutation may convert subtilisin into an amino peptidase for the same reasons substitutions of Leu126 would be expected to produce that result.

The Pro129 residue is likely to restrict the conformational freedom of the sequence 126-133, residues which may play a major role in determining P-1 specificity. Replacing Pro may introduce more flexibility thereby broadening the range of binding capabilities of such mutants.

The side chain of Lys213 is located within the S-3 subsite. All of the amino acids within the 213-215 segment are also considered to be important to substrate specificity. Accordingly, altered P-3 substrate specificity is expected upon mutation of this residue.

The Tyr214 residue does not interact with substrate but is positioned such that it could affect the conformation of the hair pin loop 204-217.

Finally, mutation of the Gly215 residue should affect the S-3′ subsite, and thereby alter P-3′ specificity.

In addition to the above substitutions of amino acids, the insertion or deletion of one or more amino acids within the external loop comprising residues 152-172 may also affect specificity. This is because these residues may play a role in the “secondary contact region” described in the model of streptomyces subtilisin inhibitor complexed with subtilisin. Hirono, et al. (1984) J. Mol. Biol. 178, 389-413. Thermitase K has a deletion in this region, which eliminates several of these “secondary contact” residues. In particular, deletion of residues 161 through 164 is expected to produce a mutant subtilisin having modified substrate specificity. In addition, a rearrangement in this area induced by the deletion should alter the position of many residues involved in substrate binding, predominantly at P-1. This, in turn, should affect overall activity against proteinaceous substrates.

The effect of deletion of residues 161 through 164 has been shown by comparing the activity of the wild type (WT) enzyme with a mutant enzyme containing this deletion as well as multiple substitutions (i.e., S153/S156/A158/G159/S160/Δ161-164/I165/S166/A169/R170). This produced the following results:

TABLE V kcat Km kcat/Km WT 50 1.4e-4 3.6e5 Deletion  8 5.0e-6 1.6e6 mutant

The WT has a kcat 6 times greater than the deletion mutant but substrate binding is 28 fold tighter by the deletion mutant. The overall efficiency of the deletion mutant is thus 4.4 times higher than the WT enzyme.

All of these above identified residues which have yet to be substituted, deleted or inserted into are presented in Table VI.

TABLE VI Substitution/Insertion/Deletion Residues His67 Ala152 Leu126 Ala153 Leu135 Gly154 Gly97 Asn155 Asp99 Gly156 Ser101 Gly157 Gly102 Gly160 Glu103 Thr158 Leu126 Ser159 Gly127 Ser161 Gly128 Ser162 Pro129 Ser163 Tyr214 Thr164 Gly215 Val165 Gly166 Tyr167 Pro168 Gly169 Lys170 Tyr171 Pro172

The mutants herein may be obtained as salts. It is clear that the ionization state of a protein will be dependent on the pH of the surrounding medium, if it is in solution, or of the solution from which it is prepared, if it is in solid form. Acidic proteins are commonly prepared as, for example, the ammonium, sodium, or potassium salts; basic proteins as the chlorides, sulfates, or phosphates. Accordingly, the present application includes both electrically neutral and salt forms of the designated carbonyl hydrolases, and the term carbonyl hydrolase referes to the organic structural backbone regardless of ionization state.

The carbonyl hydrolase mutants are particularly useful in the food processing and cleaning arts. The carbonyl hydrolases, including mutants, are produced by fermentation as described herein and recovered by suitable techniques. See for example K. Anstrup, 1974, Industrial Aspects of Biochemistry, ed. B. Spencer pp. 23-46. They are formulated with detergents or other surfactants in accord with methods known per se for use in industrial processes, especially laundry. In the latter case the enzymes are combined with detergents, builders, bleach and/or flourescent whitening agents as is known in the art for proteolytic enzymes. Suitable detergents include linear alkyl benzene sulfonates, alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linear alcohol. The compositions may be formulated in granular or liquid form. See for example U.S. Pat. Nos. 3,623,957; 4,404,128; 4,381,247; 4,404,115; 4,318,818; 4,261,868; 4,242,219; 4,142,999; 4,111,855; 4,011,169; 4,090,973; 3,985,686; 3,790,482; 3,749,671; 3,560,392; 3,558,498; and 3,557,002.

The following disclosure is intended to serve as a representation of embodiments herein, and should not be construed as limiting the scope of this application. These specific examples disclose the construction of certain of the above identified mutants. The construction of the other mutants, however, is apparent from the disclosure herein and that presented in EPO Publication No. 0130756.

Glossary of Experimental Manipulations

In order to simplify the Examples certain frequently occurring methods will be referenced by shorthand phrases.

Plasmids are designated by a small p proceeded and/or followed by capital letters and/or numbers. The starting plasmids herein are commercially available, are available on an unrestricted basis, or can be constructed from such available plasmids in accord with published procedures.

“Klenow treatment” refers to the process of filling a recessed 3′ end of double stranded DNA with deoxyribonucleotides complementary to the nucleotides making up the protruding 5′ end of the DNA strand or exonucleolytic removal of a protruding 3′ end of a double stranded DNA fragment. This process is usually used to fill in a recessed end resulting from a restriction enzyme cleavage of DNA. This creates a blunt or flush end, as may be required for further ligations. Treatment with Klenow is accomplished by reacting (generally for 15 minutes at 15° C.) the appropriate complementary deoxyribonucleotides with the DNA to be filled in under the catalytic activity (usually 10 units) of the Klenow fragment of E. coli DNA polymerase I (“Klenow”). Klenow and the other reagents needed are commercially available. The procedure has been published extensively. See for example T. Maniatis, et al., 1982, Molecular Cloning, pp. 107-108.

“Digestion” of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific is called a restriction site. “Partial” digestion refers to incomplete digestion by restriction enzyme, i.e., conditions are chosen that result in cleavage of some but not all of the sites for a given restriction endonuclease in a DNA substrate. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements as established by the enzyme suppliers were used. Restriction enzymes commonly are designated by abbreviations composed of a capital letter followed by other letters and then, generally, a number representing the microorganism from which each restriction enzyme originally was obtained. In general, about 1 μg of plasmid or DNA gragment is used with about 1 unit of enzyme in about 20 μof buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After incubation, protein is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme infrequently is followed with bacterial alkaline phosphatase hydrolysis of the terminal 5′ phosphates to prevent the two restriction cleaved ends of a DNA fragment from “circularizing” or forming a closed loop that would impede insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5′ terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional (T. Maniatis, et al., Id., pp. 133-134).

“Recovery” or “isolation” of a given fragment of DNA from a restriction digest means separation of the digest on 6 percent polyacrylamide gel electrophoresis, identification of the fragment of interest by molecular weight (using DNA fragments of known molecular weight as markers), removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example, see R. Lawn, et al., 1981, “Nucleic Acids Res.” 9:6103-6114, and D. Goeddel, et al., 1980, “Nucleic Acids Res.” 8:4057.

“Southern Analysis” is a method by which the presence of DNA sequences in a digest or DNA-containing composition is confirmed by hybridization to a known, labelled oligonucleotide or DNA fragment. For the purposes herein, Southern analysis shall mean separation of digests on 1 percent agarose and depurination as described by G. Wahl, et al., 1979, “Proc. Nat. Acad. Sci. U.S.A.” 76:3683-3687, transfer to nitrocellulose by the method of E. Southern, 1975, “J. Mol. Biol.” 98:503-517, and hybridization as described by T. Maniatis, et al., 1978, “cell” 15:687-701.

“Transformation” means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integrant. Unless otherwise stated, the method used herein for transformation of E. coli is the CaCl₂ method of Mandel, et al., 1970, “J. Mol. Biol.” 53:154, and for Bacillus, the method of Anagnostopolous, et al., 1961, “J. Bact.” 81:741-746.

“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (T. Maniatis, et al., Id., p. 146). Unless otherwise stated, ligation was accomplished using known buffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated. Plasmids from the transformants were prepared, analyzed by restriction mapping and/or sequenced by the method of Messing, et al., 1981, “Nucleic Acids Res.”, 9:309.

“Preparation” of DNA from transformants means isolating plasmid DNA from microbial culture. Unless otherwise stated, the alkaline/SDS method of Maniatis, et al., Id., p. 90, was used.

“Oligonucleotides” are short length single or double stranded polydeoxynucleotides which were chemically synthesized by the method of Crea, et al., 1980,

“Nucleic Acids Res.” 8:2331-2348 (except that mesitylene nitrotriazole was used as a condensing agent) and then purified on polyacrylamide gels.

All mutant plasmids were transformed (Anagnostopoulos, C., et al. (1961) J. Bacteriol. 81, 741-746) into BG2036 (Yang, M. (1984) J. Bacteriol. 160, 15-21) to express mutant subtilisins as described (Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521).

All literature citations are expressly incorporated by reference.

The following is presented by way of example and is :not to be construed as a limitation to the scope of the invention.

EXAMPLE 1

Identification of Peracid Oxidizable Residues of Subtilisin Q222 and L222

The activity of naturally-occurring subtilisin is rapidly reduced up to 85% by a series of oxidants. One of the most characterized modification is the conversion of Met222 to met-sulfoxide in the presence of hydrogen peroxide. Stauffer, C. E., et al. (1969) J. Biol. Chem. 244, 5333-5338. This defect has been eliminated by substituting a variety of non-oxidizable amino acids into this position by site-directed mutagenesis of the B. amyloliguifaciens enzyme, thereby confering enhanced stability to hydrogen peroxide. See EPO Publication No. 0130756 and Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518. However, as shown in FIGS. 6A and 6B, organic peracid oxidants can still inactivate the mutant enzymes Met222L and Met222Q (L222 and Q222). This example describes the identification of peracid oxidizable sites in 222 substituted mutant subtilisins.

The first step was to determine the type of amino acid involved in peracid oxidation. Except under drastic conditions (Means, G. E., et al. (1971) Chemical Modifications of Proteins, Holden-Day, S. F., CA, pp. 160-162), organic peracids modify only methionine and tryptophan in subtilisin. In order to rule out tryptophan as a candidate, difference spectra of the enzyme over the 250 nm to 350 nm range were determined during an inactivation titration employing the reagent, diperdodecanoic acid (DPDA) as the oxidant. Despite quantitative inactivation of the enzyme, no change in absorbance over this wavelength range was noted as shown in FIGS. 7A and 7B. Oxidation of tryptophan would be expected to result in marked changes over this region. Fontana, A., et al. (1980) Methods in Peptide and Protein Sequence Analysis (C. Birr ed.) Elsevier, New York, p. 309. The absence of tryptophan modification implied oxidation of one or more of the remaining methionines of B. amyloliguefaciens subtilisin. See FIG. 1.

To confirm this result the recombinant subtilisin Met222F was cleaved with cyanogen bromide (CNBr) both before and after oxidation by DPDA. The peptides produced by CNBr cleavage were analyzed on high resolution SDS-pyridine peptide gels (SPG).

Subtilisin Met222F (F222) was oxidized in the following manner. Purified F222 was resuspended in 0.1 M sodium borate pH 9.5 at 10 mg/ml and was added to a final concentration of 26 diperdodecanoic acid (DPDA) at 26 mg/ml was added to produce an effective active oxygen concentration of 30 ppm. The sample was incubated for at least 30 minutes at room temperature and then quenched with. 0.1 volume of 1 M Tris pH 8.6 buffer to produce a final concentration of 0.1 M Tris pH 8.6). 3 mM phenylmethylsulfonyl fluoride (PMSF) was added and 2.5 ml of the sample was applied to a Pharmacia PD10 column equilibrated in 10 mM sodium phosphate pH 6.2, 1 mM PMSF. 3.5 ml of 10 mM sodium phosphate pH6.2, 1 mM PMSF was applied and the eluant collected. The sample was assumed to be at 7 mg/ml based on the observation that a 2.5 ml sample of untreated F222 at 10 mg/ml in phosphate buffer when treated with PMSF and desalted in the same manner on a Pharmacia PD10 column produced a concentration of about 7 mg/ml.

F222 and DPDA oxidized F222 were precipitated with 9 volumes of acetone at −20° C. For 100 ug of protein, the acetone mixture was vortexed and centrifuged in a Fischer tabletop centrifuge for 10 minutes. The pellets were washed once with 0.5 ml acetone and then dried. The sample was carefully resuspended at 10 mg/ml in 8M urea in 88% formic acid and allowed to sit for 5 minutes. An equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein) and the samples incubated for 2 hours at room temperature in the dark. Prior to gel electrophoresis, the samples were lyophilized for 3-4 hours in a Spin Vac (Savant Instruments) and the pellets were resuspended at 2-5 mg/ml in sample buffer (1% pyridine, 5% NaDodSO₄, 5% glycerol and bromophenol blue) and disassociated at 95° C. for 3 minutes.

The samples were electrophoresed on discontinuous polyacrylamide gels as described by Kyte and Rodriguez (Kyte, J., et al. (1983) Anal. Bioch. 133, 515-522).

The gels were stained using the Pharmacia silver staining technique (Sammons, D. W., et al. (1981) Electrophoresis 2 135-141).

The results of this experiment are shown in FIG. 8. As can be seen, F222 treated with CNBr only gives nine resolved bands on SPG. However, when F222 is also treated with DPDA prior to cleavage, bands X, 7 and 9 disappear whereas bands 5 and 6 are greatly increased in intensity.

In order to determine which of the methionines were effected, each of the CNBr peptides was isolated by reversed phase HPLC and further characterized. The buffer system in both Solvent A (aqueous) and Solvent B (organic) for all HPLC separations was 0.05% TEA-TFA. Solutions were prepared by adding equal volumes of neat triethylamine and neat trifloroacetic acid to the solvent. Programs for the gradients were generated on a Waters Systems Controller. In all cases unless noted, solvent A consisted of 0.05% TEA-TFA in H20, solvent B was 0.05% TEA-TFA in 1-propanol, and the flow rate was 0.5 ml/minute.

For HPLC analysis, two injections of 1 mg enzyme digest were used. Three samples were acetone precipitated, washed and dried as above. The dried 1 mg samples were resuspended at 10 mg/ml in 8M urea, 88% formic acid; an equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein). After incubation for 2 hours in the dark at room temperature, the samples were desalted on a 0.8 cm×7 cm column of Tris Acryl GF05 coarse resin (IBF, Paris, France) equilibrated with 40% solvent B, 60% solvent A. 200 ul samples were applied at a flow rate of 1 ml a minute and 1.0-1.2 ml collected by monitoring the absorbance at 280 nm. Prior to injection on the HPLC, each desalted sample was diluted with 3 volumes of solvent A. The samples were injected at 1.0 ml/min (2 minutes) and the flow then adjusted to 0.5 ml/min (100% A). After 2 minutes, a linear gradient to 60% B at 1.0% B/min was initiated. From each 1 mg run, the pooled peaks were sampled (50 ul) and analyzed by gel electrophoresis as described above.

Each polypeptide isolated by reversed phase HPLC was further analyzed for homogeneity by SPG. The position of each peptide on the known gene sequence (Wells, J. A., et al. (1983) Nucleic Acids Res. 11 7911-7924) was obtained through a combination of amino acid compositional analysis and, where needed, amino terminal sequencing.

Prior to such analysis the following peptides were to rechromatographed.

1. CNBr Peptides from F222 Not Treated With DPDA

Peptide 5 was subjected to two additional reversed phase separations. The 10 cm C4 column was equilibrated to 80%A/20%B and the pooled sample applied and washed for 2 minutes. Next an 0.5% ml B/min gradient was initiated. Fractions from this separation were again rerun, this time on the 25 cm C4 column, and employing 0.05% TEA-TFA in acetonitrile/1-propanol (1:1) for solvent B. The gradient was identical to the one just described.

Peptide “X” was subjected to one additional separation after the initial chromatography. The sample was applied and washed for 2 minutes at 0.5 ml/min (100%A), and a 0.5% ml B/min gradient was initiated.

Peptides 7 and 9 were rechromatographed in a similar manner to the first rerun of peptide 5.

Peptide 8 was purified to homogeneity after the initial separation.

2. CNBr Peptides from DPDA Oxidized F222:

Peptides 5 and 6 from a CNBr digest of the oxidized F222 were purified in the same manner as peptide 5 from the untreated enzyme.

Amino acid compositional analysis was obtained as follows. Samples (˜nM each amino acid) were dried, hydrolyzed in vacuo with 100 ul 6N HCl at 106° C. for 24 hours and then dried in a Speed Vac. The samples were analyzed on a Beckmann 6300 AA analyzer employing ninhydrin detection.

Amino terminal sequence data was obtained as previously described (Rodriguez, H., et al. (1984) Anal. Biochem. 134, 538-547).

The results are shown in Table VII and FIG. 9.

TABLE VII Amino and COOH terminii of CNBr fragments Terminus and Method Fragment amino, method COOH, method X  1, sequence  50, composition 9  51, sequence 119, composition 7 125, sequence 199, composition 8 200, sequence 275, composition 5ox  1, sequence 119, composition 6ox 120, composition 199, composition

Peptides 5ox and 6ox refer to peptides 5 and 6 isolated from CNBr digests of the oxidized protein where their respective levels are enhanced.

From the data in Table VII and the comparison of SPG tracks for the oxidized and native protein digests in FIG. 8, it is apparent that (1) Met50 is oxidized leading to the loss of peptides X and 9 and the appearance of 5; and (2) Met124 is also oxidized leading to the loss of peptide 7 and the accumulation of peptide 6. Thus oxidation of B. amyloliguifaciens subtilisin with the peracid, diperdocecanoic acid leads to the specific oxidation of methionines 50 and 124.

EXAMPLE 2

Substitution at Met50 and Met124 in Subtilisin Met222Q

The choice of amino acid for substitution at Met50 was based on the available sequence data for subtilisins from B. licheniformis (Smith, E. C., et al. (1968) J. Biol. Chem. 243, 2184-2191), B.DY (Nedkov, P., et al. (1983) Hoppe Sayler's Z. Physiol. Chem. 364 1537-1540), B. amylosacchariticus (Markland, F. S., et al. (1967) J. Biol. Chem. 242 5198-5211) and B. subtilis (Stahl, M. L., et al. (1984) J. Bacteriol. 158, 411-418). In all cases, position 50 is a phenylalanine. See FIG. 5. Therefore, Phe50 was chosen for construction.

At position 124, all known subtilisins possess a methionine. See FIG. 5. Molecular modelling of the x-ray derived protein structure was therefore required to determine the most probable candidates for substitution. From all 19 candidates, isoleucine and leucine were chosen as the best residues to employ. In order to test whether or not modification at one site but not both was sufficient to increase oxidative stability, all possible combinations were built on the Q222 backbone (F50/Q222, I124/Q222, F50/I124/Q222).

A. Construction of Mutations Between Codons 45 and 50

All manipulations for cassette mutagenesis were carried out on pS4.5 using methods disclosed in EPO Publication No. 0130756 and Wells, J. A., et al, (1985) Gene 34, 315-323. The pΔ50 in FIG. 10, line 4, mutations was produced using the mutagenesis primer shown in FIG. 10, line 6, and employed an approach designated as restriction-purification which is described below. Wells, J. A., et al. (1986) Phil. Trans. R. Soc. Lond. A (in press). Briefly, a M13 template containing the subtilisin gene, M13mp11-SUBT was used for heteroduplex synthesis (Adelman, et al (1983), DNA 2, 183-193). Following transfection of JM101 (ATCC 33876), the 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was subcloned from M13mp11 SUBT rf into a recipient vector fragment of pBS42 the construction of which is described in EPO Publication No. 0130756. To enrich for the mutant sequence (pΔ50, line 4), the resulting plasmid pool was digested with KpnI, and linear molecules were purified by polyacrylamide gel electrophoresis. Linear molecules were ligated back to a circular form, and transformed into E. coli MM294 cells (ATCC 31446). Isolated plasmids were screened by restriction analysis for the KpnI site. KpnI⁺ plasmids were sequenced and confirmed the pΔ50 sequence. Asterisks in FIG. 11 indicate the bases that are mutated from the wid type sequence (line 4). pΔ50 (line 4) was cut with StuI and EcoRI and the 0.5 Kb fragment containing the 5′ half of the subtilisin gene was purified (fragment 1). pΔ50 (line 4) was digested with KPnI and EcoRI and the 4.0 Kb fragment containing the 3′ half of the subtilisin gene and vector sequences was purified (fragment 2). Fragments 1 and 2 (line 5), and duplex DNA cassettes coding for mutations desired (shaded sequence, line 6) were mixed in a molar ratio of 1:1:10, respectively. For the particular construction of this example the DNA cassette contained the triplet TTT for codon 50 which encodes Phe. This plasmid was designated pF-50. The mutant subtilisin was designated F-50.

B. Construction of Mutation Between Codons 122 and 127

The procedure of Example 2A was followed in substantial detail except that the mutagenesis primer of FIG. 11, line 7 was used and restriction-purification for the EcoRV site in pΔ124 was used. In addition, the DNA cassette (shaded sequence, FIG. 11, line 6) contained the triplet ATT for codon 124 which encodes Ile and CTT for Leu. Those plasmids which contained the substitution of Ile for Met124were designeated pI124. The mutant subtilisin was designated I124.

C. Construction of Various F50/I124/Q222 Multiple Mutants

The triple mutant, F50/I124/Q222, was constructed from a three-way ligation in which each fragment contained one of the three mutations. The single mutant Q222 (pQ222) was prepared by cassette mutagenesis as described in EPO Publication No. 0130756. The F50 mutation was contained on a 2.2 kb AvaII to PvuII fragment from pF50; the I124 mutation was contained on a 260 bp PvuII to AvaII fragment from pI124; and the Q222 mutation was contained on 2.7 kb AvaIl to AvaII fragment from pQ222. The three fragments were ligated together and transformed into E. coli MM294 cells. Restriction analysis of plasmids from isolated transformants confirmed the construction. To analyze the final construction it was convenient that the AvaII site at position 798 in the wild-type subtilisin gene was eliminated by the I124 construction.

The F50/Q222 and I124/Q222 mutants were constructed in a similar manner except that the appropriate fragment from pS4.5 was used for the final construction.

D. Oxidative Stability of 0222 Mutants

The above mutants were analyzed for stability to peracid oxidation. As shown in FIG. 12, upon incubation with diperdodecanoic acid (protein 2 mg/mL, oxidant 75 ppm[0]), both the I124/Q222 and the F50/I124/Q222 are completely stable whereas the F50/Q222 and the Q222 are inactivated. This indicates that conversion of M124 to I124 in subtilisin Q222 is sufficient to confer resistance to organic peracid oxidants.

EXAMPLE 3

Subtilisin Mutants Having Altered Substrate Specificity-Hydrophobic Substitutions at Residues 166

Subtilisin contains an extended binding cleft which is hydrophobic in character. A conserved glycine at residue 166 was replaced with twelve non-ionic amino acids which can project their side-chains into the S-1 subsite. These mutants were constructed to determine the effect of changes in size and hydrophobicity on the binding of various substrates.

A. Kinetics for Hydrolysis of Substrates Having Altered P-1 Amino Acids by Subtilisin BPN′ from B. amyloliguefaciens

Wild-type subtilisin was purified from B. subtilis culture supernatants expressing the B. amyloliguefaciens subtilisin gene (Wells, J. A., et al. (1983) Nucleic Acids Res. 11, 7911-7925) as previously described (Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521). Details of the synthesis of tetrapeptide substrates having the form succinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide (where X is the P1 amino acid) are described by DelMar, E. G., et al. (1979) Anal. Biochem. 99, 316-320. Kinetic parameters, Km(M) and kcat(s⁻¹) were measured using a modified progress curve analysis (Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521). Briefly, plots of rate versus product concentration were fit to the differential form of the rate equation using a non-linear regression algorithm. Errors in scat and Km for all values reported are less than five percent. The various substrates in Table VIII are ranged in order of decreasing hydrophobicity. Nozaki, Y. (1971), J. Biol. Chem. 246, 2211-2217; Tanford C. (1978) Science 200, 1012).

TABLE VIII P1 substrate kcat/Km Amino Acid kcat (S⁻¹) 1/Km (M⁻¹) (s−¹M − 1) Phe 50 7,100 360,000 Tyr 28 40,000  1,100,000   Leu 24 3,100  75,000 Met 13 9,400 120,000 His 7.9 1,600  13,000 Ala 1.9 5,500  11,000 Gly 0.003 8,300    21 Gln 3.2 2,200  7,100 Ser 2.8 1,500  4,200 Glu 0.54   32    16

The ratio of kcat/Km (also referred to as catalytic efficienty) is the apparent second order rate constant for the conversion of free enzyme plus substrate (E+S) to enzyme plus products (E+P) (Jencks, W. P., Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287). The log (kcat/Km) is proportional to transition state binding energy, ΔG_(T) ^(≠). A plot of the log kcat/Km versus the hydrophobicity of the P1 side-chain (FIG. 14) shows a strong correlation (r=0.98), with the exception of the glycine substrate which shows evidence for non-productive binding. These data show that relative differences between transition-state binding energies can be accounted for by differences in P-1 side-chain hydrophobicity. When the transition-state binding energies are calculated for these substrates and plotted versus their respective side-chain bydrophobicities, the line slope is 1.2 (not shown). A slope greater than unity, as is also the case for chymotrypsin (Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Harper, J. W., et al. (1984) Biochemistry, 23, 2995-3002), suggests that the P1 binding cleft is more hydrophobic than ethanol or dioxane solvents that were used to empirically determine the hydrophobicity of amino acids (Nozaki, Y., et al. J. Biol. Chem. (1971) 246, 2211-2217; Tanford, C. (1978) Science 200, 1012).

For amide hydrolysis by subtilisin BPN′, kcat can be interpreted as the acylation rate constant and Km as the dissociation constant, for the Michaelis complex (E·S), Ks. Gutfreund, H., et al (1956) Biochem. J. 63, 656. The fact that the log kcat, as well as log 1/Km, correlates with substrate hydrophobicity is consistent with proposals (Robertus, J. D., et al. (1972) Biochemistry 11, 2439-2449; Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303) that during the acylation step the P-1 side-chain moves deeper into the hydrophobic cleft as the substrate advances from the Michaelis complex (E·S) to the tetrahedral transition-state complex (E·S^(≠)). However, these data can also be interpreted as the hydrophobicity of the P1 side-chain effecting the orientation, and thus the susceptibility of the scissile peptide bond to nucleophilic attack by the hydroxyl group of the catalytic Ser221.

The dependence of kcat/Km on P-1 side chain hydrophobicity suggested that the kcat/Km for hydrophobic substrates may be increased by increasing the hydrophobicity of the S-1 binding subsite. To test this hypothesis, hydrophobic amino acid substitutions of Gly166 were produced.

Since hydrophobicity of aliphatic side-chains is directly proportional to side-chain surface area (Rose, G. D., et al. (1985) Science 229, 834-838; Reynolds, J. A., et al. (1974) Proc. Natl. Acad. Sci. USA 71, 2825-2927), increasing the hydrophobicity in the S-1 subsite may also sterically hinder binding of larger substrates. Because of difficulties in predicting the relative importance of these two opposing effects, we elected to generate twelve non-charged mutations at position 166 to determine the resulting specificities against non-charged substrates of varied size and hydrophobicity.

B. Cassette Mutagenesis of the P1 Binding Cleft

The preparation of mutant subtilisims containing the substitution of the hydrophobic amino acids Ala, Val and Phe into residue 166 has been described in EPO Publication No. 0130756. The same method was used to produce the remaining hydrophobic mutants at residue 166. In applying this method, two unique and silent restriction sites were introduced in the subtilisin genes to closely flank the target codon 166. As can be seen in FIG. 13, the wild type sequence (line 1) was altered by site-directed mutagenesis in M13 using the indicated 37mer mutagenesis primer, to introduce a 13 bp delection (dashedline) and unique SacI and XmaI sites (underlined sequences) that closely flank codon 166. The subtilisin gene fragment was subcloned back into the E. coli-B. subtilis shuttle plasmid, pBS42, giving the plasmid pΔ166 (FIG. 13, line 2). pΔ166 was cut open with SacI and XmaI, and gapped linear molecules were purified (FIG. 13, line 3). Pools of synthetic oligonucleotides containing the mutation of interest were annealed to give duplex DNA cassettes that were ligated into gapped pΔ166 (underlined and overlined sequences in FIG. 13, line 4). This construction restored the coding sequence except over position 166 (NNN; line 4). Mutant sequences were confirmed by dideoxy sequencing. Asterisks denote sequence changes from the wild type sequence. Plasmids containing each mutant B. amyloliguefaciens subtilisin gene were expressed at roughly equivalent levels in a protease deficient strain of B. subtilis, BG2036 as previously described. EPO Publication No. 0130756; Yang, M., et al. (1984) J. Bacteriol. 160, 15-21; Estell, D. A., et al (1985) J. Biol. Chem. 260, 6518-6521.

C. Narrowing Substrate Specificity by Steric Hindrance

To probe the change in substrate specificity caused by steric alterations in the S-1 subsite, position 166 mutants were kinetically analyzed versus P1 substrates of increasing size (i.e., Ala, Met, Phe and Tyr). Ratios of kcat/Km are presented in log form in FIG. 15 to allow direct comparisons of transition-state binding energies between various enzyme-substrate pairs.

According to transition state theory, the free enery difference between the free enzyme plus substrate (E+S) and the transition state complex (E.S^(≠)) can be calculated from equation (1),

^(Δ) G _(T) ^(≠) =−RTln kcat/Km+RT ln kT/h  (1)

in which kcat is the turnover number, Km is the Michaelis constant, R is the gas constant, T is the temperature, k is Boltzmann's constant, and h is Planck's constant. Specificity differences are ezpressed quantitatively as differences between transition state binding energies (i.e., ΔΔG_(t) ^(≠)) and can be calculated from equation (2).

^(ΔΔ) G _(T) ^(≠) =−RT ln (kcat/Km)_(A)/(kcat/Km)_(B)  (2)

A and B represent either two different substrates assayed againt the same enzyme, or two mutant enzymes assayed against the same substrate.

As can be seen from FIG. 15A, as the size of the side-chain at position 166 increases the substrate preference shifts from large to small P-1 side-chains. Enlarging the side-chain at position 166 causes kcat/Km to decrease in proportion to the size of the P-1 substrate side-chain (e.g., from Gly166 (wild-type) through W166, the kcat/Km for the Tyr substrate is decreased most followed in order by the Phe, Met and Ala P-1 substrates).

Specific steric changes in the position 166 side-chain, such as he presence of a β-hydroxyl group, β- or γ-aliphatic branching, cause large decreases in kcat/Km for larger P1 substrates. Introducing a β-hydroxyl group in going from A166 (FIG. 15A) to S166 (FIG. 15B), causes an 8 fold and 4 fold reduction in kcat/Km for Phe and Tyr substrates, respectively, while the values for Ala and Met substrates are unchanged. Producing a β-branched structure, in going from S166 to T166, results in a drop of 14 and 4 fold in kcat/Km for Phe and Tyr, respectively. These differences are slightly magnified for V166 which is slightly larger and isosteric with T166. Enlarging the β-branched substituents from V166 to I166 causes a lowering of kcat/Km between two and six fold toward Met, Phe and Tyr substrates. Inserting a γ-branched structure, by replacing M166 (FIG. 15A) with L166 (FIG. 15B), produces a 5 fold and 18 fold decrease in kcat/Km for Phe and Tyr substrates, respectively. Aliphatic γ-branched appears to induce less steric hindrance toward the Phe P-1 substrate than β-branching, as evidenced by the 100 fold decrease in kcat/Km for the Phe substrate in going from L166 to I166.

Reductions in kcat/Km resulting from increases in side chain size in the S-1 subsite, or specific structural features such as β- and γ-branching, are quantitatively illustrated in FIG. 16. The kcat/Km values for the position 166 mutants determined for the Ala, Met, Phe, and Tyr P-1 substrates (top panel through bottom panel, respectively), are plotted versus the position 166 side-chain volumes (Chothia, C. (1984) Ann. Rev. Biochem. 53, 537-572). Catalytic efficiency for the Ala substrate reaches a maximum for I166, and for the Met substrate it reaches a maximum between V166 and L166. The Phe substrate shows a broad kcat/Km peak but is optimal with A166. Here, the β-branched position 166 substitutions form a line that is parallel to, but roughly 50 fold lower in kcat/Km than side-chains of similar size (i.e., C166 versus T166, L166 versus I166. The Tyr substrate is most efficiently utilized by wild type enzyme (Gly166), and there is a steady decrease as one proceeds to large position 166 side-chains. The β-branched and γ-branched substitutions form a parallel line below the other non-charged substitutions of similar molecular volume.

The optimal substitution at position 166 decreases in volume with increasing volume of the P1 substrate (i.e., I166/Ala substrate, L166/Met substrate, A166/Phe substrate, Gly166/Tyr substrate. The combined volumes for these optimal pairs may approximate the volume for productive binding in the S-1 subsite. For the optimal pairs, Gly166/Tyr substrate, A166/Phe substrate-, L166/Met substrate, V166/Met substrate, and I166/Ala substrate, the combined volumes are 266, 295, 313, 339 and 261 A³, respectively. Subtracting the volume of the peptide backbone from each pair (i.e., two times the volume of glycine), an average side-chain volume of 160±32A³ for productive binding can be calculated.

The effect of volume, in excess to the productive binding volume, on the drop in transition-state binding energy can be estimated from the Tyr substrate curve (bottom panel, FIG. 16), because these data, and modeling studies (FIG. 2), suggest that any substitution beyond glycine causes steric repulsion. A best-fit line drawn to all the data (r=0.87) gives a slope indicating a loss of roughly 3 kcal/mol in transition state binding energy per 100A³ of excess volume. (100A³ is approximately the size of a leucyl side-chain.)

D. Enhanced Catalytic Efficiency Correlates with Increasing Hydrophobicity of the Position 166 Substitution

Substantial increases in kcat/Km occur with enlargement of the position 166 side-chain, except for the Tyr P-1 substrate (FIG. 16). For example, kcat/Km increases in progressing from Gly166 to I166 for the Ala substrate (net of ten-fold), from Gly166 to L166 for the Met substrate (net of ten-fold) and from Gly166 to A166 for the Phe substrate (net of two-fold). The increases in kcat/Km cannot be entirely explained by the attractive terms in the van der Waals potential energy function because of their strong distance dependence (1/r⁶) and because of the weak nature of these attractive forces (Jencks, W. P., Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Levitt, M. (1976) J. Mol. Biol. 104, 59-107). For example, Levitt (Levitt, M. (1976) J. Mol. Biol. 104, 59-107) has calculated that the van der Waals attraction between two methionyl residues would produce a maximal interaction energy of roughly −0.2 kcal/mol. This energy would translate to only 1.4 fold increase in kcat/Km.

The increases of catalytic efficiency caused by side-chain substitutions at position 166 are better accounted for by increases in the hydrophobicity of the S-1 subsite. The increase kcat/Km observed for the Ala and Met substrates with increasing position 166 side-chain size would be expected, because hydrophobicity is roughly proportional to side-chain surface area (Rose, G. D., et al. (1985) Science 229, 834-838; Reynolds, J. A., et al. (1974) Proc. Natl. Acad. Sci. USA 71, 2825-2927).

Another example that can be interpreted as a hydrophobic effect is seen when comparing kcat/Km for isosteric substitutions that differ in hydrophobicity such as S166 and C166 (FIG. 16). Cysteine is considerably more hydrophobic than serine (−1.0 versus +0.3 kcal/mol) (Nozaki, Y., et al. (1971) J. Biol. Chem. 246, 2211-2217; Tanford, C. (1978) Science 200, 1012). The difference in hydrophobicity correlates with the observation that C166 becomes more efficient relative to Ser166 as the hydrophobicity of the substrates increases (i.e., Ala<Met<Tye<Phe). Steric hindrance cannot explain these differences because serine is considerably smaller than cysteine (99 versus 118A³). Paul, I. C., Chemistry of the—SH Group (ed. S. Patai, Wiley Interscience, New York, 1974) pp. 111-149.

E. Production of an Elastase-Like Specificity in Subtilisin

The I166 mutation illustrates particularly well that large changes in specificity can be produced by altering the structure and hydrophobicity of the S-1 subsite by a single mutation (FIG. 17). Progressing through the small hydrophobic substrates, a maximal specificity improvement over wild type occurs for the Val substrate (16 fold in kcat/Km). As the substrate side chain size increases, these enhancements shrink to near unity (i.e., Leu and His substrates). The I166 enzyme becomes poorer against larger aromatic substrates of increasing size (e.g., I166 is over 1,000 fold worse against the Tyr substrate than is Gly166). We interpret the increase in catalytic efficiency toward the small hydrophobic substrates for I166 compared to Gly166 to the greater hydrophobicity of isoluecine (i.e., −1.8 kcal/mol versus 0). Nozaki,

Y., et al. (1971) J. Biol. Chem. 246, 2211-2217; Tanford, C. (1978) Science 200, 1012. The decrease in catalytic efficiency toward the very large substrates for I166 versus Gly166 is attributed to steric repulsion.

The specificity differences between Gly166 and I166 are similar to the specificity differences between chymotrypsin and the evolutionary relative, elastase (Harper, J. W., et al (1984) Biochemistry 23, 2995-3002). In elastase, the bulky amino acids, Thr and Val, block access to the P-1 binding site for large hydrophobic substrates that are preferred by chymotrypsin. In addition, the catalytic efficiencies toward small hydrophobic substrates are greater for elastase than for chymotrypsin as we obeseve for I166 versus Gly166 in subtilisin.

EXAMPLE 4

Substitution of Ionic Amino Acids for Gly166

The construction of subtilisin mutants containing the substitution of the ionic amino acids Asp, Asn, Gln, Lys and Ang are disclosed in EPO Publication No. 0130756. In addition, a limited analysis of substrate specificity was presented therein. The present example describes the construction of the mutant subtilisin containing Glu at position 166 (E166) and presents some of the specificity data on these mutants. Further data on position 166 and 156 single and double mutants will be presented infra.

pΔ166, described in Example 3, was digested with SacI and XmaI. The double strand DNA cassette (underlined and overlined) of line 4 in FIG. 13 contained the triplet GAA for the codon 166 to encode the replacement of Glu for Gly166. This mutant plasmid designated pQ166 was propagated in BG2036 as described. This mutant subtilisin, together with the other mutants containing ionic substituent amino acids at residue 166, were isolated as described and further analyzed for variations in substrate specificity.

Each of these mutants was analyzed with the tetrapeptide substrates, succinyl-L-AlaL-AlaProL-X-p-nitroanilide, where X was Phe, Ala and Glu.

The results of this analysis are shown in Table IX.

TABLE IX P-1 Substrate (kcat/Km × 10⁻⁴) Position 166 Phe Ala Glu Gly (wild type) 36.0 1.4 0.002 Asp (D)  0.5 0.4 <0.001 Glu (E)  3.5 0.4 <0.001 Asn (N) 18.0 1.2 0.004 Gln (Q) 57.0 2.6 0.002 Lys (K) 52.0 2.8 1.2 Arg (R) 42.0 5.0 0.08

These results indicate that charged amino acid substitutions at Gly166 have improved catalytic efficiencies (kcat/Km) for oppositely charged P-1 substrates (as much as 500 fold) and poorer catalytic efficiency for like charged P-1 substrates.

EXAMPLE 5

Substitution of Glycine at Position 169

The substitution of Gly169 in B. amyloliguefaciens subtilisin with Ala and Ser is described in EPO Publication No. 0130756. The same method was used to make the remaining 17 mutants containing all other substituent amino acids for position 169.

The construction protocol is summarized in FIG. 18. The overscored and underscored double stranded DNA cassettes used contained the following triplet encoding the substitution of the indicated amino acid at residue 169.

GCT A TGT C GAT D GAA E TTC F GGC G CAC H ATC I AAA K CTT L ATG M AAC N CCT p CAA Q AGA R AGC S ACA T GTT V TGG W TAC Y

Each of the plasmids containing a substituted Gly169 was designated pX169, where X represents the substituent amino acid. The mutant subtilisins were simialrly designated.

Two of the above mutant subtilisins, A169 and S169, were analyzed for substrate specificity against synthetic substrates containing Phe, Leu, Ala and Arg in the P-1 position. The following results are shown in Table X.

TABLE X Effect of Serine and Alanine Mutations at Position 169 on P-1 Substrate Specificity P-1 Substrate (kcat/Km × 10⁻⁴) Position 169 Phe Leu Ala Arg Gly (wild type) 40 10 1 0.4 A169 120  20 1 0.9 S169 50 10 1 0.6

These results indicate that substitutions of Ala and Ser at Gly169 have remarkably similar catalytic efficiencies against a range of P-1 substrates compared to their position 166 counterparts. This is probably because position 169 is at the bottom of the P-1 specificity subsite.

EXAMPLE 6

Substitution at Position 104

Tyr104 has been substituted with Ala, His, Leu, Met and Ser. The method used was a modification of the site directed mutagenesis method. According to the protocol of FIG. 19, a primer (shaded in line 4) introduced a unique HindIII site and a frame shift mutation at codon 104. Restriction-purification for the unique HindIII site facilitated the isolation of the mutant sequence (line 4). Restriction-selection against this HindIII site using pimers in line 5 was used to obtain position 104 mutants.

The following triplets were used in the primers of FIG. 19, line 5 for the 104 codon which substituted the following amino acids.

GCT Ala TTC Phe ATG Met CCT Pro CTT Leu ACA Thr AGC Ser TGG Trp CAC His TAC Tyr CAA Gln GTT Val GAA Glu AGA Arg GGC Gly AAC Asn ATC Ile GAT Asp AAA Lys TGT Cys

The following substrates were used to analyze the substrate specificity of these mutants to give the indicated results in Table XI.

TABLE XI kcat Km Kcat/Km Substrate WT H104 WT H104 WT H104 sAAPFpNA 50.0 22.0 1.4e − 4 7.1e − 4 3.6e5 3.1e4 sAAPApNA 3.2 2.0 2.3e − 4 1.9e − 3 1.4e4   1e3 sFAPFpNA 26.0 38.0 1.8e − 4 4.1e − 4 1.5e5 9.1e4 sFAPApNA 0.32 2.4 7.3e − 5 1.5e − 4 4.4e3 1.6e4

From these data it is clear that the substitution of His for Tyr at position 104 produces an enzyme which is more efficient (higher kcat/Km) when Phe is at the P-4 substrate position than when Ala is at the P-4 substrate position.

EXAMPLE 7

Substitution of Ala152

Ala152 has been substituted by Gly and Ser to determine the effect of such substitutions on substrate specificity.

The wild type DNA sequence was mutated by the V152/P153 primer (FIG. 20, line 4) using the above restriction-purification approach for the new KpnI site. Other mutant primers (shaded sequences FIG. 20; S152, line 5 and G152, line 6) mutated the new KpnI site away and such mutants were isolated using the restriction-selection procedure as described above for loss of the KpnI site.

The results of these substitutions for the above synthetic substrates containing the P-1 amino acids Phe, Leu and Ala are shown in Table XII.

TABLE XII P-1 Substrate (kcat/Km × 10⁻⁴) Position 152 Phe Leu Ala Gly (G) 0.2 0.4 <0.04 Ala (wild type) 40.0  10.0  1.0 Ser (S) 1.0 0.5 0.2

These results indicate that, in contrast to positions 166 and 169, replacement of Ala152 with Ser or Gly causes a dramatic reduction in catalytic efficiencies across all substrates tested. This suggests Ala152, at the top of the S-1 subsite, may be the optimal amino acid because Ser and Gly are homologous Ala substitutes.

EXAMPLE 8

Substitution at Position 156

Mutants containing the substitution of Ser and Gln for Glu156 have been constructed according to the overall method depicted in FIG. 21. This method was designed to facilitate the construciton of multiple mutants at position 156 and 166 as will be described hereinafter. However, by regenerating the wild type Gly166, single mutations at Glu156 were obtained.

The plasmid pΔ166 is already depicted in line 2 of FIG. 13. The synthetic oligonucleotides at the top right of FIG. 21 represent the same DNA cassettes depicted in line 4 of FIG. 13. The plasmid p166 in FIG. 21 thus represents the mutant plasmids of Examples 3 and 4. In this particular example, p166 contains the wild type Gly166.

Construction of position 156 single mutants were prepared by ligation of the three fragments (1-3) indicated at the bottom of FIG. 21. Fragment 3, containing the carboxy-terminal portion of the subtilisin gene including the wild type position 166 codon, was isolated as a 610 bp SacI-BamHI fragment. Fragment 1 contained the vector sequences, as well as the amino-terminal sequences of the subtilisin gene through codon 151. To produce fragment 1, a unique KPnI site at codon 152 was introduced into the wild type subtilisin sequence from pS4.5. Site-directed mutagenesis in M13 employed a primer having the sequence 5′-TA-GTC-GTT-GCG-GTA-CCC-GGT-AAC-GAA-3′ to produce the mutation. Enrichment for the mutant sequence was accomplished by restriction with KpnI, purification and self ligation. The mutant sequence containing the KpnI site was confirmed by direct plasmid sequencing to give pV-152. pV-152 (˜1 μg) was digested with KpnI and treated with 2 units of DNA polymerase I large fragment (Klenow fragment from Boeringer-Mannheim) plus 50 μM deoxynucleotide triphosphates at 37° C. for 30 min. This created a blunt end that terminated with codon 151. The DNA was extracted with 1:1 volumes phenol and CHCl₃ and DNA in the aqueous phase was precipitated by addition of 0.1 volumes 5M ammonium acetate and two volumes ethanol. After centrifugation and washing the DNA pellet with 70% ethanol, the DNA was lyophilized. DNA was digested with BamHI and the 4.6 kb piece (fragment 1) was purified by acrylamide gel electrophoresis followed by electroelution. Fragment 2 was a duplex synthetic DNA cassette which when ligated with fragments 1 and 3 properly restored the coding sequence except at codon 156. The top strand was synthesized to contain a glutamine codon, and the complementary bottom strand coded for serine at 156. Ligation of heterophosphorylated cassettes leads to a large and favorable bias for the phosphorylated over the non-phosphorylated oligonucleotide sequence in the final segrated plasmid product. Therefore, to obtain Q156 the top strand was phosphorylated, and annealed to the non-phosphorylated bottom strand prior to ligation. Similarly, to obtain S156 the bottom strand was phosphorylated and annealed to the non-phosphorylated top strand. Mutant sequences were isolated after ligation and transformation, and were confirmed by restriction analysis and DNA sequencing as before. To express variant subtilisins, plasmids were transformed into a subtilisin-neutral protease deletion mutant of B. subtilis, BG2036, as previously described. Cultures were fermented in shake flasks for 24 h at 37° C. In LB media containing 12.5 mg/mL chloraphenicol and subtilisin was purified from culture supernatants as described. Purity of subtilisin was greater than 95% as judged by SDS PAGE.

These mutant plasmids designated pS156 and pQ156 and mutant subtilisins designated S156 and Q156 were analyzed with the above synthetic substrates where P-1 comprised the amino acids Glu, Gln, Met and Lys. The results of this analyses are presented in Example 9.

EXAMPLE 9

Multiple Mutants With Altered Substrate Specificity—Substitution at Positions 156 and 166

Single substitutions of position 166 are described in Examples 3 and 4. Example 8 describes single substitutions at position 156 as well as the protocol of FIG. 21 whereby various double mautants comprising the substitution of various amino acids at positions 156 and 166 can be made. This example describes the construction and substrate specificity of subtilisin containing substitutions at position 156 and 166 and summarized some of the data for single and double mutants at positions 156 and 166 with various substrates.

K166 is a common replacement amino acid in the 156/166 mutants described herein. The replacement of Lys for Gly166 was achieved by using the synthetic DNA cassette at the top right of FIG. 21 which contained the triplet AAA for NNN. This produced fragment 2 with Lys substituting for Gly166.

The 156 substituents were Gln and Ser. The Gln and Ser substitutions at Gly156 are contained within fragment 3 (bottom right FIG. 21).

The multiple mutants were produced by combining fragments 1, 2 and 3 as described in Example 8. The mutants Q156/K166 and S156/K166 were selectively generated by differential phosphorylation as described. Alternatively, the double 156/166 mutants, c.f. Q156/K166 and S156/K166, were prepared by ligation of the 4.6 kb SacI-BamHI fragment from the relevant p156 plasmid containing the 0.6 kb SacI-BamHI fragment from the relevant p166 plasmid.

These mutants, the single mutant K166, and the S156 and Q156 mutants of Example 8 were analyzed for substitute specificity against synthetic polypeptides containing Phe or Glu as the P-1 substrate residue. The results are presented in Table XIII.

TABLE XIII Sub- strate kcat/Km Enzymes P-1 (mutant) Compared^((b)) Residue kcat Km kcat/Km kcat/Km(wt) Glu-156/ Phe 50.00 1.4 × 10⁻⁴ 3.6 × 10⁵ (1) Gly-166 (WT) Glu 0.54 3.4 × 10⁻² 1.6 × 10¹ (1) Lys-166 Phe 20.00 4.0 × 10⁻⁵ 5.2 × 10⁵ 1.4 Glu 0.70 5.6 × 10⁻⁵ 1.2 × 10⁴ 750 Gln-156/Lys-166 Phe 30.00 1.9 × 10⁻⁵ 1.6 × 10⁶ 4.4 Glu 1.60 3.1 × 10⁻⁵ 5.0 × 10⁴ 3100 Ser-156/Lys-166 Phe 30.00 1.8 × 10⁻⁵ 1.6 × 10⁶ 4.4 Glu 0.60 3.9 × 10⁻⁵ 1.6 × 10⁴ 1000 Ser-156 Phe 34.00 4.7 × 10⁻⁵ 7.3 × 10⁵ 2.0 Glu 0.40 1.8 × 10⁻³ 1.1 × 10² 6.9 Glu-156 Phe 48.00 4.5 × 10⁻⁵ 1.1 × 10⁶ 3.1 Glu 0.90 3.3 × 10⁻³ 2.7 × 10² 17

As can be seen in Table XIV, either of these single mutations improve enzyme performance upon substrates with glutamate at the P-1 enzyme binding site. When these single mutations were combined, the resulting multiple enzyme mutants are better than either parent. These single or multiple mutations also alter the relative pH activity profiles of the enzymes as shown in FIG. 23.

To isolate the contribution of electrostatics to substrate specificity from other chemical binding forces, these various single and double mutants were analyzed for their ability to bind and cleave synthetic substrates containing Glu, Gln, Met and Lys as the P-1 substrate amino acid. This permitted comparisons between side-chains that were more sterically similar but differed in charge (e.g., Glu versus Gln, Lys versus Met). Similarly, mutant enzymes were assayed against homologous P-1 substrates that were most sterically similar but differed in charge (Table XIV).

TABLE XIV Kinetics of Position 156/166 Subtilisins Determined for Different P1 Substrates Enzyme Net P-1 Substrate log kcat/Km (log 1/Km)^((c)) Position^((a)) Charge^((b)) Glu Gln Met Lys 156 166 Glu Asp −2 n.d. 3.02 (2.56) 3.93 (2.74) 4.23 (3.00) Glu Glu −2 n.d. 3.06 (2.91) 3.86 (3.28) 4.48 (3.69) Glu Asn −1 1.62 (2.22) 3.85 (3.14) 4.99 (3.85) 4.15 (2.88) Glu Gln −1 1.20 (2.12) 4.36 (3.64) 5.43 (4.36) 4.10 (3.15) Gln Asp −1 1.30 (1.79) 3.40 (3.08) 4.94 (3.87) 4.41 (3.22) Ser Asp −1 1.23 (2.13) 3.41 (3.09) 4.67 (3.68) 4.24 (3.07) Glu Met −1 1.20 (2.30) 3.89 (3.19) 5.64 (4.83) 4.70 (3.89) Glu Ala −1 n.d. 4.34 (3.55) 5.65 (4.46) 4.90 (3.24) Glu Gly(wt) −1 1.20 (1.47) 3.85 (3.35) 5.07 (3.97) 4.60 (3.13) Gln Gly 0 2.42 (2.48) 4.53 (3.81) 5.77 (4.61) 3.76 (2.82) Ser Gly 0 2.31 (2.73) 4.09 (3.68) 5.61 (4.55) 3.46 (2.74) Gln Asn 0 2.04 (2.72) 4.51 (3.76) 5.79 (4.66) 3.75 (2.74) Ser Asn 0 1.91 (2.78) 4.57 (3.82) 5.72 (4.64) 3.68 (2.80) Glu Arg 0 2.91 (3.30) 4.26 (3.50) 5.32 (4.22) 3.19 (2.80) Glu Lys 0 4.09 (4.25) 4.70 (3.88) 6.15 (4.45) 4.23 (2.93) Gln Lys +1 4.70 (4.50) 4.64 (3.68) 5.97 (4.68) 3.23 (2.75) Ser Lys +1 4.21 (4.40) 4.84 (3.94) 6.16 (4.90) 3.73 (2.84) Maximum difference: log kcat/Km (log 1/Km)^((d)) 3.5 (3.0) 1.8 −1.3 (−1.0) Footnotes to Table XIV: ^((a)) B. subtilis, BG 2036, expressing indicated variant subtilisin were fermented and enzymes purified as previously described (Estell, et al. (1985) J. Biol. Chem. 260, 6518-6521). Wild type subtilisin is indicated (wt) containing Glu156 and Gly166. ^((b))Net charge in the P-1 binding site is defined as the sum of charges from positions 156 and 166 at pH 8.6. ^((c))Values for kcat(s⁻¹) and Km(M) were measured in 0.1M Tris pH 8.6 at 25° C. as previously described³ against P-1 substrates having the form succinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide, where X is the indicated P-1 amino acid. Values for log 1/Km are shown inside parentheses. All errors in determination of kcat/Km and 1/Km are below 5%. ^((d))Because values for Glu156/Asp166(D166) are too small to determine accurately, the maximum difference taken for GluP-1 substrate is limited to a charge range of +1 to −1 charge change. n.d. =not determined

The kcat/Km ratios shown are the second order rate constants for the conversion of substrate to product, and represent the catalytic efficiency of the enzyme. These ratios are presented in logarithmic form to scale the data, and because log kcat/Km is proportional to the lowering of transition-state activation energy (ΔG_(T)). Mutations at position 156 and 166 produce changes in catalytic efficiency toward Glu, Gln, Met and Lys P-1 substrates of 3100, 60, 200 and 20 fold, respectively. Making the P-1 binding-site more positively charged (e.g., compare Gln156/Lys166 (Q156/K166) versus Glu156/Met166 (Glu156/M166)dramatically increased kcat/Km toward the Glu P-1 substrate (up to 3100 fold), and decreased the catalytic efficiency toward the Lys P-1 substrate (up to 10 fold). In addition, the results show that the catalytic efficiency of wild type enzyme can be greatly improved toward any of the four P-1 substrates by mutagenesis of the P-1 binding site.

The changes in kcat/Km are caused predominantly by changes in 1/Km. Because 1/Km is approximately equal to 1/Ks, the enzyme-substrate association constant, the mutations primarily cause a change in substrate binding. These mutations produce smaller effects on kcat that run parallel to the effects on 1/Km. The changes in kcat suggest either an alteration in binding in the P-1 binding site in going from the Michaelis-complex E·S) to the transition-state complex (E−S≠) as previously proposed (Robertus, J. D., et al. (1972) Biochemistry 11, 2439-2449; Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), or change in the position of the scissile peptide bond over the catalytic serine in the E·S complex.

Changes in substrate preference that arise from changes in the net charge in the P-1 binding site show trands that are best accounted for by electrostatic effects (FIG. 28). As the P-1 binding cleft becomes more positively charged, the average catalytic efficiency increases much more for the Glu P-1 substrate than for its neutral and isosteric P-1 homolog, Gln (FIG. 28A). Furthermore, at the positive extreme both substrates have nearly identical catalytic efficiencies.

In contrast, as the P-1 site becomes more positively charged the catalytic efficiency toward the Lys P-1 substrate decreases, and diverges sharply from its neutral and isosteric homolog, Met (FIG. 28B). The similar and parallel upward trend seen with increasing positive charge for the Met and Glu P-1 substrates probably results from the fact that all the substrates are succinylated on their amino-terminal end, and thus carry a formal negative charge.

The trends observed in log kcat/Km are dominated by changes in the Km term (FIGS. 28C and 28D). As the pocket becomes more positively charged, the log 1/Km values converge for Glu and Gln P-1 substrates (FIG. 28C), and diverge for Lys and Met P-1 substrates (FIG. 28D). Although less pronounced effects are seen in log kcat, the effects of P-1 charge on log kcat parallel those seen in log 1/Km and become larger as the P-1 pocket becomes more positively charged. This may result from the fact that the transition-state is a tetrahedral anion, and a net positive charge in the enzyme may serve to provide some added stabilization to the transition-state.

The effect of the change in P-1 binding-site charge on substrate preference can be estimated from the differences in slopes between the charged and neutral isosteric P-1 substrates (FIG. 28B). The average change in substrate preference (Δlog kcat/Km) between charged and neutral isosteric substrates increases roughly 10-fold as the complementary charge or the enzyme increases (Table XV). When comparing Glu versus Lys, this difference is 100-fold and the change in substrate preference appears predominantly in the Km term.

TABLE XV Differential Effect on Binding Site Charge on log kcat/Km or (log 1/Km) for P-1 Substrates that Differ in Charge^((a)) Change in P-1 Binding Δlog kcat/Km (Δlog 1/Km) Site Charge ^((b)) GluGln MetLys GluLys −2 to −1 n.d. 1.2 (1.2) n.d. −1 to 0   0.7 (0.6) 1.3 (0.8) 2.1 (1.4)   0 to +1 1.5 (1.3) 0.5 (0.3) 2.0 (1.5) Avg. change in 1.1 (1.0) 1.0 (0.8) 2.1 (1.5) log kcat/Km or (log 1/Km) per unit charge change ^((a))The difference in the slopes of curves were taken between the P-1 substrates over the charge interval given for log (k(cat/Km) (Figure 3A, B) and (log 1/Km) (Figure 3D, D). Values represent the differential effect a charge change has in distinguishing the substrates that are compared. ^((b))Charge in P-1 binding site is defined as the sum of charges from positions 156 and 166.

The free energy of electrostatic interactions in the structure and energetics of salt-bridge formation depends on the distance between the charges and the microscopic dielectric of the media. To dissect these structural and microenvironmental effects, the energies involved in specific salt-bridges were evaluated. In addition to the possible salt-bridges shown (FIGS. 29A and 29B), reasonable salt-bridges can be built between a Lys P-1 substrate and Asp at position 166, and between a Glu P-1 substrate and a Lys at position 166 (not shown). Although only one of these structures is confirmed by X-ray crystalography (Poulos, T. L., et al. (1976) J. Mol. Biol. 257 1097-1103), all models have favorable torsion angles (Sielecki, A. R., et al. (1979) J. Mol. Biol. 134, 781-804), and do not introduce unfavorable van der Waals contacts.

The change in charged P-1 substrate preference brought about by formation of the model salt-bridges above are shown in Table XVI.

TABLE XVI Effect of Salt Bridge Formation Between Enzyme and Substrate on P1 Substrate preference^((a)) Change Substrate^((d)) in Substrate Enzyme P-1 Preference Preference Enzymes Compared^((b)) Position Substrates Δlog (kcat/Km) ΔΔlog (kcat/Km) 1 2 Changed Compared 1 2 (1-2) Glu156/Asp166 Gln156/Asp166 156 LysMet +0.30 −0.53 0.83 Glu156/Asp166 Gln156/Asn166 156 LysMet −0.84 −2.04 1.20 Glu156/Gly166 Gln156/Gly166 156 LysMet −0.47 −2.10 1.63 Glu156/ Gln156/Lys166 156 LysMet −1.92 −2.74 0.82 Lsy-166 Ave ΔΔlog (kcat/Km) 1.10 ± 0.3 Glu156/Asp166 Glu156/Asn166 166 LysMet +0.30 −0.84 1.14 Glu156/Glu166 Glu156/Glu166 166 LysMet +0.62 −1.33 1.95 Gln156/Asp166 Gln156/Asn166 166 LysMet −0.53 −2.04 1.51 Ser156/Asp166 Ser156/Asn166 166 LysMet −0.43 −2.04 1.61 Glu156/Lys166 Glu156/Met166 166 GluGln −0.63 −2.69 2/06 Ave ΔΔlog (kcat/Km) 1.70 ± 0.3 Footnotes to Table XVI: ^((a))Molecular modeling shows it is possible to form a salt bridge between the indicated charged P-1 substrate and a complementary charge in the P-1 binding site of the enzyme at the indicated position changed. ^((b))Enzymes compared have sterically similar amino acid substitutions that differ in charge at the indicated position. ^((c))The P-1 substrates compared are structurally similar but differ in charge. The charged P-1 substrate is complementary to the charge change at the position indicated between enzymes 1 and 2. ^((d))Date from Table XIV was used to compute the difference in log (kcat/Km) between the charged and the non-charged P-1 substrate (i.e., the substrate preference). The substrate preference is shown separately for enzyme 1 and 2. ^((e))The difference in substrate preference between enzyme 1 (more highly charged) and enzyme 2 (more neutral) represents the rate change accompanying the electrostatic interaction.

The difference between catalytic efficiencies (i.e., Δlog kcat/Km) for the charged and neutral P-1 substrates (e.g., Lys minus Met or Glu minus Gin) give the substrate preference for each enzyme. The change in substrate preference (ΔΔlog kcat/Km) between the charged and more neutral enzyme homologs (e.g., Glu156/Gly166 minus Gln156 (Q156)/Gly166) reflects the change in catalytic efficiency that may be attributed solely to electrostatic effects.

These results show that the average change in substrate preference is considerably greater when electrostatic substitutions are produced at position 166 (50-fold in kcat/Km) versus position 156 (12-fold in kcat/Km). From these ΔΔ log kcat/Km values, an average change in transition-state stabilization energy can be calculated of −1.5 and −2.4 kcal/mol for substitutions at positions 156 and 166, respectively. This should represent the stabilization energy contributed from a favorable electrostatic interaction for the binding of free enzyme and substrate to form the transition-state complex.

At least three factors can contribute to the higher transition-state binding energies for electrostatic interactions at position 166. These include: (i) smaller charge separation for salt-bridges at position 166; (ii) more stable side-chain geometries for salt-bridges at position 166; and (iii) microscopic dielectric constants at positions 166.

It is unreasonable to expect all of the energy difference to be due to shorter salt bridges at position 166, because these would have to be 1.6 times shorter than at position 156 for which crystalographic data (Mathews, D. A., et al. (1975) J. Biol. Chem. 250, 7120-7126) indicate are optimally formed. Furthermore, molecular models of salt-bridges appear as structurally reasonable at 156 as at 166.

The binding energies may be more easily explained as differences in the microscopic dielectric constants at position 156 and 166. Assuming a salt-bridge distance of 3A, ˜2.7A), the calculated dielectric constant at position 156 would be 72 (ΔGe=Z₁Z₂/rD where Z is the charge on particle 1 and 2, r is the charge separation, and D is the dielectric constant). This corresponds closely with the dielectric constant of 78 for water at this temperature, and qualitatively fits with the fact that position 156 is located on the surface of the enzyme, and is freely exposed to solvent. A calculated dielectric constant for a salt-bridge at position 166 is 45, and fits with the fact that position 166 is more buried and less accessible to solvent. Furthermore, our estimate, based on the hydrophobicity, of the P-1 binding site, indicates that P-1 binding site has an overall dielectric constant close to that of ethanol (D=25).

A large number of mutant comparisons is necessary to show a statistically significant difference between salt-bridges at positive 156 and 166 because there is considerable variation in ΔΔ log kcat/Km for different mutant comparisons at the same position. The change in susbtrate preference from putative salt-bridges at position 156 varies from six to 40-fold in kcat/Km, and those at position 166 vary 14 to 120 fold.

In addition to variation produced by factors mentioned above, it is possible that the P-1 side chains are not binding in the same ways between the enzymes compared, even though the comparisons are nearly isosteric in each case. For example, the Lys P-1 substrate side chain may contact Glu156 in Glu156/Asp166 (Glu156/D166) and Asp166 in Gln156/Asp166 (Q156/D166). Thus, one salt-bridge may be substitued for another. It is also possible that complementary charges within the P-1 binding site, e.g., Glu156/Lys166 (Glu156/K166), can form an intramolecular salt-bridge so that the charged side-chains are not free to interact independently with the substrate. Given these caveats it is remarkable that greater variation in substrate preference is not seen by electrostatic substitutions at each position.

EXAMPLE 10

Substitutions at Position 217

Tyr217 has been substituted by all other 19 amino acids. Cassette mutagenesis as described in EPO publication No. 0130756 was used according to the protocol of FIG. 22. The EcoRV restriction site was used for restriction-purification of p^(Δ)217.

Since this position is involved in substrate binding, mutations here affect kinetic parameters of the enzyme. An example is the substitution of Leu for Tyr at position 217. For the substrate sAAPFpNa, this mutant has a kcat of 277 5′ and a Km of 4.7×10⁻⁴ with a kcat/Km ratio of 6×10⁵. This represents a 5.5-fold increase in kcat with a 3-fold increase in Km over the wild type enzyme.

In addition, replacement of Tyr217 by Lys, Arg, Phe or Leu results in mutant enzymes which are more stable at pH of about 9-11 than the WT enzyme. Conversely, replacement of Tyr217 by Asp, Glu, Gly or Pro results in enzymes which are less stable at pH of about 9-11 than the WT enzyme.

EXAMPLE 11

Multiple Mutants Having Altered Thermal Stability

B. amyloliguefacien subtilisin does not contain any cysteine residues. Thus, any attempt to produce thermal stability by Cys cross-linkage required the substitution of more than one amino acid in subtilisin with Cys. The following subtilisin residues were multiply substituted with cysteine:

Thr22/Ser87

Ser24/Ser87

Mutagenesis of Ser24 to Cys was carried out with a 5′ phosphorylated oligonucleotide primer having the sequence

(Asterisks show the location of mismatches and the underlined sequence shows the position of the altered Sau3A site.) The B. amyloliguefaciens subtilisin gene on a 1.5 kb EcoRI-BAMHI fragment from pS4.5 was cloned into M13mp11 and single stranded DNA was isolated. This template (M13mp11SUBT) was double primed with the 5′ phosphorylated M13 universal sequencing primer and the mutagenesis primer. Adelman, et al. (1983) DNA 2, 183-193. The heteroduplex was transfected into competent JM101 cells and plaques were probed for the mutant sequence (Zoller, M. J., et al. (1982) Nucleic Acid Res. 10, 6487-6500; Wallace, et al. (1981) Nucleic Acid Res. 9, 3647-3656) using a tetramethylammonium chloride hybridization protocol (Wood, et al. (1985) Proc. Natl. Acad. Sci. USA 82, 1585-1588). The Ser87 to Cys mutation was prepared in a similar fashion using a 5′ phosphorylated primer having the sequence

(The asterisk indicates the position of the mismatch and the underlined sequence shows the position of a new MstI site.) The C24 and C87 mutations were obtained at a frequency of one and two percent, respectively. Mutant sequences were confirmed by dideoxy sequencing in M13.

Mutagenesis of Tyr21/Thr22 to A21/C22 was carried out with a 5′ phosphorylated oligonucleotide primer having the sequence

(The asterisks show mismatches to the wild type sequence and the underlined sequence shows the position of an altered Sau3A site.) Manipulations for heteroduplex synthesis were identical to those described for C24. Because direct cloning of the heteroduplex DNA fragment can yield increased frequencies of mutagenesis, the EcoRI-BamHI subtilisin fragment was purified and ligated into pBS42. E. coli MM 294 cells were transformed with the ligation mixture and plasmid DNA was purified from isolated transformants. Plasmid DNA was screened for the loss of the Sau3A site at codon 23 that was eliminated by the mutagenesis primer. Two out of 16 plasmid preparations had lost the wild type Sau3A site. The mutant sequence was confirmed by dideoxy sequencing in M13.

Double mutants, C22/C87 and C24/c87, were constructed by ligating fragments sharing a common ClaI site that separated the single parent cystine codons. Specifically, the 500 bp EcoRI-ClaI fragment containing the 5′ portion of the subtilisin gene (including codons 22 and 24) was ligated with the 4.7 kb ClaI-EcoRI fragment that contained the 3′ portion of the subtilisin gene (including codon 87) plus pBS42 vector sequence. E. coli MM 294. was transformed with ligation mixtures and plasmid DNA was purified from individual transformants. Double-cysteine plasmid constructions were identified by restriction site markers originating from the parent cysteine mutants (i.e., C22 and C24, Sau3A minus; Cys87, MstI plus). Plasmids from E. coli were transformed into B. subtilis BG2036. The thermal stability of these mutants as compared to wild type subtilisin are presented in FIG. 30 and Tables XVII and XVIII.

TABLE XVII Effect of DTT on the Half-Time of Autolytic Inactivation of Wild-Type and Disulfide Mutants of Subtilisin* t_(1/2) −DDT +DTT Enzyme min −DTT/+DTT Wild-type 95 85 1.1 C22/C87 44 25 1.8 C24/C87 92 62 1.5 *Purified enzymes were either treated or not treated with 25 mM DTT and dialyzed with or without 10 mM DTT in 2 mM CaCl₂, 50 mM Tris (pH 7.5) for 14 hr. at 4° C. Enzyme concentrations were adjusted to 80 μl aliquots were quenched on ice and assayed for residual activity. Half-times for autolytic inactivation were determined from semi-log plots of log₁₀ (residual activity) versus time. These plots were linear for over 90% of the inactivation.

TABLE XVIII Effect of Mutations in Subtilisin on the Half-Time of Autolytic Inactivation at 58° C.* t_(1/2) Enzyme min Wild-type 120 C22  22 C24 120 C87 104 C22/C87  43 C24/C87 115 *Half-times for autolytic inactivation were determined for wild-type and mutant subtilisins as described in the legend to Table III. Unpurified and non-reduced enzymes were used directly from B. subtilis culture supernatants.

It has been demonstrated that double-cysteine mutants of subtilisin are efficiently secreted and that disulfide bonds are formed in vivo in B. subtilis (date not shown). The introduction of disulfide bonds in subtilisin extends upon previous work in dihydrofolate reductase and T4 lysozyme (Perry, L. J., et al. (1984) Science 226, 555-557), where single cysteines were introduced near pre-existing cysteines and disulfides were oxidized in vitro. Analyses of physical properties of the subtilisin disulfides, unlike the T4 lysozyme disulfide (Perry, L. J., et al. (1986) Biochemistry, in press), were not complicated by the presence of free cysteines other than those involved in disulfide formation. Because most naturally occuring disulfides occur in secreted proteins, subtilisin is an excellent model system to identify the structural requirements for in vitro formation of stable disulfide bonds in secreted proteins.

Thermal Stability and Autolytic Stability

The data presented here do not address reverisble thermostability of subtilisin directly because of complications arising from autolysis and aggregation. For example, studies monitoring the change in the circular dichroic eliptcity at 220 nm versus temperature of phenylmethanesulfonyl fluoride-inhibited subtilisin show typical melt profiles that are coincident with the autolysis curves. However, at the end of thermal melt, SDS-PAGE shows that >90% of the subtilisin is autolyzed. Moreover, Brown and Schleich (Brown, M. F., et al. (1975) Biochemistry 14, 3069-3074) have shown that diisopropylfluorophos-phate-inhibited subtilisin irreversibly aggregates in denaturants, which precludes reversible denaturation studies. Thus, until these problems are overcome, subtilisin is not an ideal system for studying the thermodynamics of protein folding.

Although there appears to be a relationship between autolytic stability and conformational stability, the disulfides introduced into subtilisin did not improve the autolytic stability of the mutant enzymes when compared to the wild-type enzyme. However, the disulfide bonds did provide a margin of autolytic stability when compared to their corresponding reduced double-cysteine enzyme. Inspection of a highly refined x-ray structure of wild-type B. amyloliguefaciens subtilisin reveals a hydrogen bond between Thr22 and Ser87. Because cysteine is a poor hydrogen donor or acceptor (Paul, I. C. (1974) in Chemistry of the —SH Group (Patai, S., ed.) pp. 111-149, Wiley Interscience, New York) weakening of 22/87 hydrogen bond may explain why the C22 and C87 single-cysteine mutant proteins are less autolytically stable than either C24 or wild-type (Table XVIII). The fact that C22 is less autolytically stable than C87 may be the result of the Tyr21A mutation (Table XVIII). Indeed, recent construction and analysis of Tyr21/C22 shows the mutant protein has an autolytic stability closer to that of C87. In summary, the C22 and C87 of single-cysteine mutations destabilize the protein toward autolysis, and disulfide bond formation increases the stability to a level less than or equal to that of wild-type enzyme.

These data suggest that the stabilizing effect of an engineered disulfide can be lowered when the parent cysteine mutations disrupt pre-existing stabilizing interactions. Similar conclusions have been drawn from reversible thermal unfolding studies of disulfide cross-linked T4 lysozyme mutants that contain destabilizing mutations. Therefore, a strategy to stbilize a protein by introduction of a disulfide bond should consider avoiding the disruption of stabilizing interactions as well as producing a disulfide with good bond geometry.

EXAMPLE 12

Multiple Mutants Containing Substitutions at Position 222 and Position 166 or 169

Double mutants 166/222 and 169/222 were prepared by ligating together (1) the 2.3 kb AcaII fragment from pS4.5 which contains the 5′ portion of the subtilisin gene and vector sequences, (2) the 200 bp AvaII fragment which contains the relevant 166 or 169 mutations from the respective 166 or 169 plasmids, and (3) the 2.2 kb AvaII fragment which contains the relevant 222 mutation 3′ and of the subtilisin genes and vector sequence from the respective p222 plasmid.

Although mutations at position 222 improve oxidation stability they also tend to increase the Km. An example is shown in Table XIX. In this case the A222 mutation was combined with the K166 mutation to give an enzyme with kcat and Km intermediate between the two parent enzymes.

TABLE XIX substrate sAAPFpNa kcat Km WT 50 1.4 × 10⁻⁴ A222 42 9.9 × 10⁻⁴ K166 21 3.7 × 10⁻⁵ K166/A222 29 2.0 × 10⁻⁴

EXAMPLE 13

Multiple Mutants Containing Substitutions at Positions 50, 156, 166, 217 and Combinations Thereof

The double mutant S156/A169 was prepared by ligation of two fragments, each containing one of the relevant mutations. The plasmid pS156 was cut with XmaI and treated with S1 nuclease to create a blunt end at codon 167. After removal of the nuclease by phenol/chloroform extraction and ethanol precipitation, the DNA was digested with BamHI and the approximately 4 kb fragment containing the vector plus the 5′ portion of the subtilisin gene through codon 167 was purified.

The pA169 plasmid was digested with KpnI and treated with DNA polymerase Klenow fragment plus 50 μM dNTPs to create a blunt end codon at codon 168. The Klenow was removed by phenol/chloroform extraction and ethanol precipitation. The DNA was digested with BamHI and the 590 bp fragment including codon 168 through the carboxy terminus of the subtilisin gene was isolated. The two fragments were then ligated to give S156/A169.

Triple and quadruple mutants were prepared by ligating together (1) the 220 bp PvuII/HaeII fragment containing the relevant 156, 166 and/or 169 mutations from the respective p156, p166 and/or p169 double of single mutant plasmid, (2) the 550 bp HaeII/BamHI fragment containing the relevant 217 mutant from the respective p217 plasmid, and (3) the 3.9 kb PvuII/BamHI fragment containing the F50 mutation and vector sequences.

The multiple mutant F50/S156/A169/L217, as well as B. amyloliguefaciens subtilisin, B. lichenformis subtilisin and the single mutant L217 were analyzed with the above synthetic polypeptides where the P-1 amino acid in the substrate was Lys, His, Ala, Gln, Tyr, Phe, Met and Leu. These results are shown in FIGS. 26 and 27.

These results show that the F50/S156/A169/L217 mutant has substrate specificity similar to that of the B. licheniformis enzyme and differs dramatically from the wild type enzyme. Although only data for the L217 mutant are shown, none of the single mutants (e.g., F50, S156 or A169) showed this effect. Although B. licheniformis differs in 88 residue positions from B. amyloliguefaciens, the combination of only these four mutations accounts for most of the differences in substrate specificity between the two enzymes.

EXAMPLE 14

Subtilisin Mutants Having Altered Alkaline Stability

A random mutagenesis technique was used to generate single and multiple mutations within the B. amyloliguefaciens subtilisin gene. Such mutants were screened for altered alkaline stability. Clones having increased (positive) alkaline stability and decreased (negative) alkaline stability were isolated and sequenced to identify the mutations within the subtilisin gene. Among the positive clones, the mutants V107 and R213 were identified. These single mutants were subsequently combined to produce the mutant V107/R213.

One of the negative clones (V50) from the random mutagenesis experiments resulted in a marked decrease in alkaline stability. Another mutant (P50) was analyzed for alkaline stability to determine the effect of a different substitution at position 50. The F50 mutant was found to have a greater alkaline stability than wild type subtilisin and when combined with the double mutant V107/R213 resulted in a mutant having an alkaline stability which reflected the aggregate of the alkaline stabilities for each of the individual mutants.

The single mutant R204 and double mutant C204/R213 were identified by alkaline screening after random cassette mutagenesis over the region from position 197 to 228. The C204/R213 mutant was thereafter modified to produce mutants containing the individual mutations C204 and R213 to determine the contribution of each of the individual mutations. Cassette mutagenesis using pooled oligonucleotides to substitute all amino acids at position 204, was utilized to determine which substitution at position 204 would maximize the increase in alkaline stability. The mutation from Lys213 to Arg was maintained constant for each of these substitutions at position 204.

A. Construction of pB0180, an E. coli-B. subtilis Shuttle Plasmid

The 2.9 kb EcoRI-BamHI fragment from pBR327 (Covarrubias, L., et al. (1981) Gene 13, 25-35) was ligated to the 3.7 kb EcoRI-BamHI fragment of pBD64 (Gryczan, T., et al. (1980) J. Bacteriol., 246-253) to give the recombinant plasmid pB0153. The unique EcoRI recognition sequence in pBD64 was eliminated by digestion with EcoRI followed by treatment with Klenow and deoxynucleotide triphosphates (Maniatis, T., et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Blunt end ligation and transformation yielded pB0154. The unique AvaI recognition sequence in pB0154 was eliminated in a similar manner to yield pB0171. pB0171 was digested with BamHI and PvuII and treated with Klenow and deoxynucleotide triphosphates to create blunt ends. The 6.4 kb fragment was purified, ligated and transformed into LE392 cells (Enquest, L. W., et al. (1977) J. Mol. Biol. 111, 97-120), to yield pB0172 which retains the unique BamHI site. To facilitate subcloning of subtilisin mutants, a unique and silent KpnI site starting at codon 166 was introduced into the subtilisin gene from pS4.5 (Wells, J. A., et al. (1983) Nucleic Acids Res., 11, 7911-7925) by site-directed mutagenesis. The KpnI+ plasmid was digested with EcoRI and treated with Klenow and deoxynucleotide triphosphates to create a blunt end. The Klenow was inactivated by heating for 20 min at 68° C., and the DNA was digested with BamHI. The 1.5 kb blunt EcoRI-BamHI fragment containing the entire subtilisin was ligated with the 5.8 kb NruI-BamHI from pB0172 to yield pB0180. The ligation of the blunt NruI end to the blunt EcoRI end recreated an EcoRI site. Proceeding clockwise around pB0180 from the EcoRI site at the 5′ end of the subtilisin gene is the unique BamHI site at the 3′ end of the subtilisin gene, the chloramphenicol and neomycin resistance genes and UB110 gram positive replication origin derived from pBD64, the ampicillin resistance gene and gram negative replication origin derived from pBR327.

B. Construction of Random Mutagenesis Library

The 1.5 kb EcoRI-BamHI fragment containing the B. amyloliguefaciens subtilisin gene (Wells et al., 1983) from pB0180 was cloned into M13mp11 to give M13mp11 SUBT essentially as previously described (Wells, J. A., et al. (1986) J. Biol. Chem., 261,6564-6570). Deoxyuridine containing template DNA was prepared according to Kunkel (Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA, 82 488-492). Uridine containing template DNA (Kunkel, 1985) was purified by CsCl density gradients (Maniatis, T. et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). A primer (AvaI) having the sequence

ending at codon-11, was used to alter the unique AvaI recognition sequence within the subtilisin gene. (The asterisk denotes the mismatches from the wild-type sequence and underlined is the altered AvaI site.) The 5′ phosphorylated AvaI primer (˜320 pmol) and ˜40 pmol (˜120 μg) of uridine containing M13mp11 SUBT template in 1.88 ml of 53 nM NaCl, 7.4 mM MgCl2 and 7.4 mM Tris.HCl (pH 7.5) were annealed by heating to 90° C. for 2 min. and cooling 15 min at 24° C. (FIG. 31). Primer extension at 24° C. was initiated by addition of 100 μL containing 1 mM in all four deoxynucleotide triphosphates, and 20 μl Klenow fragment (5 units/1). The extension reaction was stopped every 15 seconds over ten min by addition of 10 μl 0.25 M EDTA (pH 8) to 50 μl aliquots of the reaction mixture. Samples were pooled, phenol chlorophorm extract and DNA was precipitated twice by addition of 2.5 vol 100% ethanol, and washed twice with 70% ethanol. The pellet was dried, and redissolved in 0.4 ml 1 mM EDTA, 10 m Tris (pH 8). Misincorporation of α-thiodeoxynucleotides onto the 3′ ends of the pool of randomly terminated template was carried out by incubating four 0.2 ml solutions each containing one-fourth of the randomly terminated template mixture (˜20 μg), 0.25 nM of a given α-thiodeoxynucleotide triphosphate, 100 units AMV polymerase, 50 mM KCL, 10 mM MgCl₂, 0.4 m dithiothreitol, and 50 mM Tris (pH 8.3) (Champoux, J. J. (1984) Genetics, 2, 454-464). After incubation at 37° C. for 90 minutes, misincorporation reactions were sealed by incubation for five minutes at 37° C. with 50 mM all four deoxynucleotide triphosphates (pH 8), and 50 units AMV polymerase. Reactions were stopped by addition of 25 mM EDTA (final), and heated at 68° C. for ten min to inactivate AMV polymerase. After ethanol precipitation and resuspension, synthesis of closed circular heteroduplexes was carried out for two days at 14° C. under the same conditions used for the timed extension reactions above, except the reactions also contained 1000 units T4 DNA ligase, 0.5 mM ATP and 1 mM β-mercaptoethanol. Simultaneous restriction of each heteroduplex pool with KpnI, BamHI, and EcoRI confirmed that the extension reactions were nearly quantitative. Heteroduplex DNA in each reaction mixture was methylated by incubation with 80 μM S-adenosylmethionine and 150 units dam methylase for 1 hour at 37° C. Methylation reactions were stopped by heating at 68° C. for 15 min. One-half of each of the four methylated heteroduplex reactions were transformed into 2.5 ml competent E. coli JM101 (Messing, J. (1979) Recombinant DNA Tech. Bull., 2, 43-48). The number of independent transformants from each of the four transformations ranged from 0.4-2.0×10⁵. After growing out phage pools, RF DNA from each of the four transformations was isolated and purified by centrifugation through CsCl density gradients. Approximately 2 μg of RF DNA from each of the four pools was digested with EcoRI, BamHI and AvaI. The 1.5 kb EcoRI-BamHI fragment (i.e., AvaI resistant) was purified on low gel temperature agarose and ligated into the 5.5 kb EcoRI-BamHI vector fragment of pBO180. The total number of independent transformants from each a-thiodeoxynucleotide misincorporation plasmid library ranged from 1.2-2.4×10⁴. The pool of plasmids from each of the four transformations was grown out in 200 ml LB media containing 12.5 μg/ml cmp and plasmid DNA was purified by centrifugation through CsCl density gradients.

C. Expression and Screening of Subtilisin Point Mutants

Plasmid DNA from each of the four misincorporation pools was transformed (Anagnostopoulos, C., et al. (1967, J. Bacteriol., 81, 741-746) into BG2036, a strain of B. subtilis deficient in extracellular protease genes (Yang, M. Y. et al. (1984) J. Bacteriol., 160, 15-21). For each transformation, 5 μg of DNA produced approximately 2.5×10⁵ independent BG2036 transformants, and liquid culture aliquots from the four libraries were stored in 10% glycerol at 70° C. Thawed aliquots of frozen cultures were plated on LB/5 μg/ml cmp/1.6% skim milk plates (Wells, J. A., et al. (1983) Nucleic Acids Res., 11, 7911-7925), and fresh colonies were arrayed onto 96-well microtiter plates containing 150 1 per well LB media plus 12.5 μg/ml cmp. After 1 h at room temperature, a replica was stamped (using a matched 96 prong stamp) onto a 132 mm BA 85 nitrocellulose filter (Schleicher and Scheull) which was layered on a 140 mm diameter LB/cmp/skim milk plate. Cells were grown about 16 h at 30° C. until halos of proteolysis were roughly 5-7 mm in diameter and filters were transferred directly to a freshly prepared agar plate at 37° C. containing only 1.6% skim milk and 50 mM sodium phosphate pH 11.5. Filters were incubated on plates for 3-6 h at 37° C. to produce halos of about 5 mm for wild-type subtilisin and were discarded. The plates were stained for 10 min at 24° C. with Coomassie blue solution (0.25% Coomassie blue (R-250) 25% ethanol) and destained with 25% ethanol, 10% acetic acid for 20 min. Zones of proteolysis appeared as blue halos on a white background on the underside of the plate and were compared to the original growth plate that was similarly stained and destained as a control. Clones were considered positive that produced proportionately larger zones of proteolysis on the high pH plates relative to the original growth plate. Negative clones gave smaller halos under alkaline conditions. Positive and negative clones were restreaked to colony purify and screened again in triplicate to confirm alkaline pH results.

D. Identification and Analysis of Mutant Subtilisins

Plasmid DNA from 5 ml overnight cultures of more alkaline active B.subtilis clones was prepared according to Birnboim and Doly (1979) except that incubation with 2 mg/ml lysozyme proceeded for 5 min at 37° C. to ensure cell lysis and an additional phenol/CHCl₃ extraction was employed to remove contaminants. The 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was ligated into M13mp11 and template DNA was prepared for DNA sequencing (Messing, J., et al. (1982) Gene, 19 269-276). Three DNA sequencing primers ending at codon 26, +951 and +155 were synthesized to match the subtilisin coding sequence. For preliminary sequence identification a single track of DNA sequence, corresponding to the dNTPaS misincorporation library from which the mutant came, was applied over the entire mature protein coding sequence (i.e., a single dideoxyguanosine sequence track was applied to identify a mutant from the dGTPas library). A complete four track of DNA sequence was performed 200 bp over the site of mutagenesis to confirm and identify the mutant sequence (Sanger, F., et al., (1980) J. Mol. Biol., 143, 161-178). Confirmed positive and negative bacilli clones were cultured in LB media containing 12.5 μg/mL cmp and purified from culture supernatants as previously described (Estell, D. A., et al. (1985) J. Biol. Chem., 260, 6518-6521). Enzymes were greater than 98% pure as analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, U. K. (1970), Nature, 227, 680-685), and protein concentrations were calculated from the absorbance at 280 nm (ε₂₈₀ ^(0.1%)=1.17, Maturbara, H., et al. (1965), J. Biol. Chem, 1125-1130).

Enzyme activity was measured with 200 μg/mL succinyl-L-AlaL-AlaL-ProL-Phep-nitroanilide (Sigma) in 0.1M Tris pH 8.6 or 0.1 M CAPS pH 10.8 at 25° C. Specific activity (μmoles product/min-mg) was calculated from the change in absorbance at 410 nm from production of p-nitroaniline with time per mg of enzyme (E410=8,480 M-1cm-1; Del Mar, E. G., et al. (1979), Anal. Biochem., 99, 316-320). Alkaline autolytic stability studies were performed on purified enzymes (200 μg/mL) in 0.1 M potassium phosphate (pH 12.0) at 37° C. At various times aliquots were assayed for residual enzyme activity (Wells, J. A., et al. (1986) J. Biol. Chem., 261, 6564-6570).

E. Results

1. Optimization and Analysis of Mutagenesis Frequency

A set of primer-template molecules that were randomly 3′-terminated over the subtilisin gene (FIG. 31) was produced by variable extension from a fixed 5′ -primer (The primer mutated a unique AvaI site at codon 11 in the subtilisin gene). This was achieved by stopping polymerase reactions with EDTA after various times of extension. The extent and distribution of duplex formation over the 1 kb subtilisin gene fragment was assessed by multiple restriction digestion (not shown). For example, production of new HinfI fragments identified when polymerase extension had proceeded past Ile110, Leu233, and Asp259 in the subtilisin gene.

Misincorporation of each dNTPas at randomly terminated 3′ ends by AMV reverse transcriptase (Zakour, R. A., et al. (1982), Nature, 295, 708-710; Zakour, R. A., et al. (1984), Nucleic Acids Res., 12, 6615-6628) used conditions previously described (Champoux, J. J., (1984), Genetics, 2, 454-464). The efficiency of each misincorporation reaction was estimated to be greater than 80% by the addition of each dNTPαs to the AvaI restriction primer, and analysis by polyacrylamide gel electrophoresis (Champoux, J. J., (1984). Misincorporations were sealed by polymerization with all four dNTP's and closed circular DNA was produced by reaction with DNA ligase.

Several manipulations were employed to maximize the yield of the mutant sequences in the heteroduplex. These included the use of a deoxyuridine containing template (Kunkel, T. A. (1985), Proc. Natl. Acad. Sci. USA, 82 488-492; Pukkila, P. J. et al. (1983), Genetics, 104, 571-582), invitro methylation of the mutagenic strand (Kramer, W. et al. (1982) Nucleic Acids Res., 10 6475-6485), and the use of AvaI restriction-selection against the wild-type template strand which contained a unique AvaI site. The separate contribution of each of these enrichment procedures to the final mutagenesis frequency was not determined, except that prior to AvaI restriction-selection roughly one-third of the segregated clones in each of the four pools still retained a wild-type AvaI site within the subtilisin gene. After AvaI restriction-selection greater than 98% of the plasmids lacked the wild-type AvaI site.

The 1.5 kb EcoRI-BamHI subtilisin gene fragment that was resistant to AvaI restriction digestion, from each of the four CsCl purified M13 RF pools was isolated on low melting agarose. The fragment was ligated in situ from the agarose with a similarly cut E. coli-B. subtilis shuttle vector, pB0180, and transformed directly into E coli LE392. Such direct ligation and transformation of DNA isolated from agarose avoided loses and allowed large numbers of recombinants to be obtained (>100,000 per μg equivalent of input M13 pool).

The frequency of mutagenesis for each of the four dNTPas misincorporation reactions was estimated from the frequency that unique restriction sites were eliminated (Table XX). The unique restriction sites chosen for this analysis, ClaI, PvuII, and KpnI, were distributed over the subtilisin gene starting at codons 35, 104, and 166, respectively. As a control, the mutagenesis frequency was determined at the PstI site located in the βlactamase gene which was outside the window of mutagenesis. Because the absolute mutagenesis frequency was close to the percentage of undigested plasmid DNA, two rounds of restriction-selection were necessary to reduce the background of surviving uncut wild-type plasmid DNA below the mutant plasmid (Table XX). The background of surviving plasmid from wild-type DNA probably represents the sum total of spontaneous mutations, uncut wild-type plasmid, plus the efficiency with which linear DNA can transform E. coli . Subtracting the frequency for unmutagenized DNA (background) from the frequency for mutant DNA, and normalizing for the window of mutagenesis sampled by a given restriction analysis (4-6 bp) provides an estimate of the mutagenesis efficiency over the entire coding sequence (_(—)1000 bp).

TABLE XX α-thiol % resis- % dNTP Restriction % resistant clones^(c) tant clones mutants misincor- Site 1st 2nd over Back- per porated^((b)) Selection round round Total ground^((d)) 1000 bp^((e)) None PstI 0.32 0.7 0.002 0 — G PstI 0.33 1.0 0.003 0.001 0.2 T PstI 0.32 <0.5 <0.002 0 0 C PstI 0.43 3.0 0.013 0.011 3 None ClaI 0.28 5 0.014 0 — G ClaI 2.26 85 1.92 1.91 380 T ClaI 0.48 31 0.15 0.14 35 C ClaI 0.55 15 0.08 0.066 17 None PvuII 0.08 29 0.023 0 — G PvuII 0.41 90 0.37 0.35 88 T PvuII 0.10 67 0.067 0.044 9 C PvuII 0.76 53 0.40 0.38 95 None KpnI 0.41 3 0.012 0 — G KpnI 0.98 35 0.34 0.33 83 T KpnI 0.36 15 0.054 0.042 8 C KpnI 1.47 26 0.38 0.37 93 ^((a))Mutagenesis frequency is estimated from the frequency for obtaining mutations that alter unique restriction sites within the mutagenized subtilisin gene (i.e., ClaI, PvuII, or KpnI) compared to mutation frequencies of the PstI site, that is outside the window of mutagenesis. ^((b))Plasmid DNA was from wild-type (none) or mutagenized by dNTPαs misincorporation as described. ^((c))Percentage of resistant clones was calculated from the fraction of clones obtained after three fold or greater over-digestion of the plasmid with the indicated restriction enzyme compared to a non-digested control. Restriction-resistant plasmid DNA from the first round was subjected to a second round of restriction-selection. The total represents the product of the fractions of resistant clones obtained from both rounds of selection and gives percentage of restriction-site mutant clones in # the original starting pool. Frequencies were derived from counting at least 20 colonies and usually greater than 100. ^((d))Percent resistant clones was calculated by subtracting the percentage of restriction-resistant clones obtained for wild-type DNA (i.e., none) from that obtained for mutant DNA. ^((e))This extrapolates from the frequency of mutation over each restriction site to the entire subtilisin gene (^(˜)1 kb). This has been normalized to the number of possible bases (4-6 bp) within each restriction site that can be mutagenized by a given misincorporation event.

From this analysis, the average percentage of subtilisin genes containing mutations that result from dGTPas, dCTPαs, or dTTPαs misincorporation was estimated to be 90, 70, and 20 percent, respectively. These high mutagenesis frequencies were generally quite variable depending upon the dNTPαs and misincorporation efficiencies at this site. Misincorporation efficiency has been reported to be both dependent on the kind of mismatch, and the context of primer (Champoux, J. J., (1984); Skinner, J. A., et al. (1986) Nucleic Acids Res., 14, 6945-6964). Biased misincorporation efficiency of dGTPαs and dCTPαs over dTTPαs has been previously observed (Shortle, D., et al. (1985), Genetics, 110, 539-555). Unlike the dGTPαs, dCTPαs, and dTTPαs libraries the efficiency of mutagenesis for the dATPαs misincorporation library could not be accurately assessed because 90% of the restriction-resistant plasmids analyzed simply lacked the subtilisin gene insert. This problem probably arose from self-ligation of the vector when the dATPαs mutagenized subtilisin gene was subcloned from M13 into pB0180. Correcting for the vector background, we estimate the mutagenesis frequency around 20 percent in the dATPαs misincorporation library. In a separate experiment (not shown), the mutagenesis efficiencies for dGTPαs and dTTPαs misincorporation were estimated to be around 50 and 30 percent, respectively, based on the frequency of reversion of an inactivating mutation at codon 169.

The location and identity of each mutation was determined by a single track of DNA sequencing corresponding to the misincorporated αthiodeoxy-nucleotide over the entire gene followed by a complete four track of DNA sequencing focused over the site of mutation. Of 14 mutants identified, the distribution was similar to that reported by Shortle and Lin (1985) except we did not observe nucleotide insertion or deletion mutations. The proportion of AG mutations was highest in the G misincorporation library, and some unexpected point mutations appeared in the dTTPas and dCTPas libraries.

2. Screening and Identification of Alkaline Stability Mutants of Subtilisin

It is possible to screen colonies producing subtilisin by halos of casein digestion (Wells, J. A. et al. (1983) Nucleic Acids Res., 11, 7911-7925). However, two problems were posed by screening colonies under high alkaline conditions (>pH 11). First, B. subtilis will not grow at high pH, and we have been unable to transform an alkylophilic strain of bacillus. This problem was overcome by adopting a replica plating strategy in which colonies were grown on filters at neutral pH to produce subtilisin and filters subsequently transferred to casein plates at pH 11.5 to assay subtilisin activity. However, at pH 11.5 the casein micells no longer formed a turbid background and thus prevented a clear observation of proteolysis halos. The problem was overcome by briefly staining the plate with Coomassie blue to amplify proteolysis zones and acidifying the plates to develop casein micell turbidity. By comparison of the halo size produced on the reference growth plate (pH 7) to the high pH plate (pH 11.5), it was possible to identify mutant subtilisins that had increased (positives) or decreased (negatives) stability under alkaline conditions.

Roughly 1000 colonies were screened from each of the four misincorporation libraries. The percentage of colonies showing a differential loss of activity at pH 11.5 versus pH 7 represented 1.4, 1.8, 1.4, and 0.6% of the total colonies screened from the thiol dGTPαs, dATPαs, dTTPαs, and dCTPαs libraries, respectively. Several of these negative clones were sequenced and all were found to contain a single base change as expected from the misincorporation library from which they came. Negative mutants included A36, E170 and V50. Two positive mutants were identified as V107 and R213. The ratio of negatives to positives was roughly 50:1.

3. Stability and Activity of Subtilisin Mutants at Alkaline pH

Subtilisin mutants were purified and their autolytic stabilities were measured by the time course of inactivation at pH 12.0 (FIGS. 32 and 33). Positive mutants identified from the screen (i.e., V107 and R213) were more resistant to alkaline induced autolytic inactivation compared to wild-type; negative mutants (i.e., E170 and V50) were less resistant. We had advantageously produced another mutant at position 50 (F50) by site-directed mutagenesis. This mutant was more stable than wild-type enzyme to alkaline autolytic inactivation (FIG. 33) At the termination of the autolysis study, SDS-PAGE analysis confirmed that each subtilisin variant had autolyzed to an extent consistent with the remaining enzyme activity.

The stabilizing effects of V107, R213, and F50 are cumulative. See Table XXI. The double mutant, V107/R213 (made by subcloning the 920 bp EcoRI-KpnI fragment of pB0180V107 into the 6.6 kb EcoRI-KPnI fragment of pB0180R213), is more stable than either single mutant. The triple mutant, F50/V107/R213 (made by subcloning the 735 bp EcoRI-PvuII fragment of pF50 (Example 2) into the 6.8 kb EcoRI-PvuII fragment of pB0180/V107, is more stable than the double mutant V107/R213 or F50. The inactivation curves show a biphasic character that becomes more pronounced the more stable the mutant analyzed. This may result from some destablizing chemical modification(s) (eg., deamidation) during the autolysis study and/or reduced stabilization caused by complete digestion of larger autolysis peptides. These alkaline autolysis studies have been repeated on separately purified enzyme batches with essentially the same results. Rates of autolysis should depend both on the conformational stability as well as the specific activity of the subtilisin variant (Wells, J. A., et al. (1986), J. Biol. Chem., 261, 6564-6570). It was therefore possible that the decreases in autolytic inactivation rates may result from decreases in specific activity of the more stable mutant under alkaline conditions. In general the opposite appears to be the case. The more stable mutants, if anything, have a relatively higher specific activity than wild-type under alkaline conditions and the less stable mutants have a relatively lower specific activity. These subtle effects on specific activity for V107/R213 and F50/V107/R213 are cumulative at both pH 8.6 and 10.8. The changes in specific activity may reflect slight differences in substrate specificity, however, it is noteworthy that only positions 170 and 107 are within 6 Å of a bound model substrate (Robertus, J. D., et al. (1972), Biochemistry 11, 2438-2449).

TABLE XXI Relationship between relative specific acitivity at pH 8.6 or 10.8 and alkaline autolytic stability Alkaline autolysis Relative specific activity half-time Enzyme pH 8.6 pH 10.8 (min) b Wild-type 100 ± 1 100 ± 3  86 Q170  46 ± 1  28 ± 2  13 V107 126 ± 3  99 ± 5 102 R213  97 ± 1 102 ± 1 115 V107/R213 116 ± 2 106 ± 3 130 V50  66 ± 4  61 ± 1  58 F50 123 ± 3 157 ± 7 131 F50/V107/ 126 ± 2 152 ± 3 168 R213 (a) Relative specific activity was the average from triplicate activity determinations divided by the wild-type value at the same pH. The average specific activity of wild-type enzyme at pH 8.6 and 10.8 was 70 μmoles/min-mg and 37 μmoles/min-mg, respectively. (b) Time to reach 50% activity was taken from FIGS. 32 and 33.

F. Random Cassette Mutagenesis of Residues 197 through 228

Plasmid pΔ222 (Wells, et al. (1985) Gene 34, 315-323) was digested with PstI and BamHI and the 0.4 kb PstI/BamHI fragment (fragment 1, see FIG. 34) purified from a polyacrylamide gel by electroelution (Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The 1.5 kb EcoRI/BamHI fragment from pS4.5 was cloned into M13mp9. Site directed mutagenesis was performed to create the A197 mutant and simultaneously insert a silent SstI site over codons 195-196. The mutant EcoRI/BamHI fragment was cloned back into pBS42. The A197 plasmid was digested with BamHI and SstI and the 5.3 kb BamHI/SstI fragment (fragment 2) was purified from low melting agarose.

Complimentary oligonucleotides were synthesized to span the region from SstI (codons 195-196) to PstI (codons 228-230). These oligodeoxynucleotides were designed to (1) restore codon 197 to the wild type, (2) re-create a silent KpnI site present in pΔ222 at codons 219-220, (3) create a silent SmaI site over codons 210-211, and (4) eliminate the PstI site over codons 228-230 (see FIG. 35). Oligodeoxynucleotides were synthesized with 2% contaminating nucleotides at each cycle of synthesis, e.g., dATP reagent was spiked with 2% dCTP, 2% dGTP, and 2% dTTP. For 97-mers, this 2% poisoning should give the following percentages of non-mutant, single mutants and double or higher mutants per strand with two or more misincorporations per complimentary strand: 14% non-mutant, 28% single mutant, and 57% with ≧2 mutations, according to the general formula $f = {{\frac{\mu^{n}}{n!}} - {\mu.}}$

where μ is the average number of mutations and n is a number class of mutations and f is the fraction of the total having that number of mutations. Complimentary oligodeoxynucleotide pools were phosphorylated and annealed (fragment 3) and then ligated at 2-fold molar excess over fragments 1 and 2 in a three-way ligation.

E. coli MM294 was transformed with the ligation reaction, the transformation pool grown up over night and the pooled plasmid DNA was isolated. This pool represented 3.4×10⁴ independent transformants. This plasmid pool was digested with PstI and then used to retransform E. coli. A second plasmid pool was prepared and used to transform B. subtilis (BG2036). Approximately 40% of the BG2036 transformants actively expressed subtilisin as judged by halo-clearing on casein plates. Several of the non-expressing transformants were sequenced and found to have insertions or deletions in the synthetic cassettes. Expressing BG2036 mutants were arrayed in microtiter dishes with 150 μl of LB/12.5 μg/mL chloramphenicol (cmp) per well, incubated at 37° C. for 3-4 hours and then stamped in duplicate onto nitrocellulose filters laid on LB 1.5% skim milk/5 μg/mL cmp plates and incubated overnight at 33° C. (until halos were approximately 4-8 mm in diameter). Filters were then lifted to stacks of filter paper saturated with 1×Tide commercial grade detergent, 50 mM Na₂CO₃, pH 11.5 and incubated at 65° C. for 90 min. Overnight growth plates were Commassie stained and destained to establish basal levels of expression. After this treatment, filters were returned to pH7/skim milk/20 μg/mL tetracycline plates and incubated at 37° C. for 4 hours to overnight.

Mutants identified by the high pH stability screen to be more alkaline stable were purified and analyzed for autolytic stability at high pH or high temperature. The double mutant C204/R213 was more stable than wild type at either high pH or high temperature (Table XXII).

This mutant was dissected into single mutant parents (C204 and R213) by cutting at the unique SmaI restriction site (FIG. 35) and either ligating wild type sequence 3′ to the SmaI site to create the single C204 mutant or ligating wild type sequence 5′ to the SmaI site to create the single R213 mutant. Of the two single parents, C204 was nearly as alkaline stable as the parent double mutant (C204/R213) and slightly more thermally stable. See Table XXII. The R213 mutant was only slightly more stable than wild type under both conditions (not shown).

Another mutant identified from the screen of the 197 to 228 random cassette mutagenesis was R204. This mutant was more stable than wild type at both high pH and high temperature but less stable than C204.

Table XXII

Stability of Subtilisin Variants

Purified enzymes (200 μg/mL) were incubated in 0.1M phosphate, pH 12 at 30° C. for alkaline autolysis, or in 2 mM CaCl₂, 50 mM MOPS, pH 7.0 at 62° C. for thermal autolysis. At various times samples were assayed for residual enzyme activity. Inactivations were roughly pseudo-first order, and t 1/2 gives the time it took to reach 50% of the starting activity in two separate experiments.

t 1/2 t 1/2 (alkaline (thermal autolysis) autolysis) Exp. Exp. Exp. Exp. Subtilisin variant #1 #2 #1 #2 wild type 30 25 20 23 F50/V107/R213 49 41 18 23 R204 35 32 24 27 C204 43 46 38 40 C204/R213 50 52 32 36 L204/R213 32 30 20 21

G. Random Mutagenesis at Codon 204

Based on the above results, codon 204 was targeted for random mutagenesis. Mutagenic DNA cassettes (for codon at 204) all contained a fixed R213 mutation which was found to slightly augment the stability of the C204 mutant.

Plasmid DNA encoding the subtilisin mutant C204/R213 was digested with SstI and EcoRI and a 1.0 kb EcoRI/SstI fragment was isolated by electro-elution from polyacrylamide gel (fragment 1, see FIG. 35).

C204/R213 was also digested with SmaI and EcoRI and the large 4.7 kb fragment, including vector sequences and the 3′ portion of coding region, was isolated from low melting agarose (fragment 2, see FIG. 36).

Fragments 1 and 2 were combined in four separate three-way ligations with heterophosphorylated fragments 3 (see FIGS. 36 and 37). This hetero-phosphorylation of synthetic duplexes should preferentially drive the phosphorylated strand into the plasmid ligation product. Four plasmid pools, corresponding to the four ligations, were restricted with SmaI in order to linearize any single cut C204/R213 present from fragment 2 isolation, thus reducing the background of C204/R213. E. coli was then re-transformed with SmaI-restricted plasmid pools to yield a second set of plasmid pools which are essentially free of C204/R213 and any non-segregated heterduplex material.

These second enriched plasmid pools were then used to transform B. subtilis (BG2036) and the resulting four mutant pools were screened for clones expressing subtilisin resistant to high pH/temperature inactivation. Mutants found positive by such a screen were further characterized and identified by sequencing.

The mutant L204/R213 was found to be slightly more stable than the wild type subtilisin. See Table XXII.

Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention. 

What is claimed is:
 1. A modified subtilisin comprising a substitution of at least one amino acid residue in the amino acid sequence of a precursor subtilisin in at least one position equivalent to Met50, Met124, or Met222 of the amino acid sequence of naturally produced Bacillus amyloliguefaciens subtilisin, wherein said modified subtilisin has altered oxidative stability in comparison to said precursor subtilisin.
 2. The modified subtilisin of claim 1 wherein said precursor subtilisin is a recombinant subtilisin.
 3. The modified subtilisin of claim 1 comprising a substitution of at least two of said amino acid residues of said precursor subtilisin.
 4. The modified subtilisin of claim 1 wherein said modified subtilisin is substantially isolated.
 5. A modified subtilisin not found in nature comprising a substitution of at least one amino acid residue in the amino acid sequence of a precursor subtilisin in at least one position equivalent to His67, Gly97, Asp99, Gly100, Ser101, Gly102, Gln103, Tyr104, Ile107, Leu126, Gly127, Gly128, Pro129, Leu135, Ala152, Ala153, Gly154, Asn155, Glu156, Gly157, Thr158, Ser159, Gly160, Ser161, Ser162, Ser163, Thr164, Val165, Gly166, Tyr167, Pro168, Gly169, Lys170, Tyr171, Pro172, Phe189, Lys213, Tyr214, Gly215, or Tyr217 of the amino acid sequence of naturally produced Bacillus amyloliguefaciens subtilisin, wherein said modified subtilisin has altered substrate specificity in comparison to said precursor subtilisin.
 6. The modified subtilisin of claim 5 wherein said precursor subtilisin is a recombinant subtilisin.
 7. The modified subtilisin of claim 5 comprising a substitution of at least two of said amino acid residues of said precursor subtilisin.
 8. The modified subtilisin of claim 5 wherein said modified subtilisin is substantially isolated.
 9. A modified subtilisin comprising a substitution of at least one amino acid residue in the amino acid sequence of a precursor subtilisin in at least one position equivalent to Tyr21, Thr22, Ser87, Asn 155, Glu156, Gly166, Gly169, Tyr217, or Met222 of the amino acid sequence of naturally produced Bacillus amyloliguefaciens subtilisin, wherein said modified subtilisin has altered catalytic activity in comparison to said precursor subtilisin.
 10. The modified subtilisin of claim 9 wherein said precursor subtilisin is a recombinant subtilisin.
 11. The modified subtilisin of claim 9 comprising a substitution of at least two of said amino acid residues of said precursor subtilisin.
 12. The modified subtilisin of claim 9 wherein said modified subtilisin is substantially isolated.
 13. A modified subtilisin comprising a substitution of at least one amino acid residue in the amino acid sequence of a precursor subtilisin in at least one position equivalent to Tyr21, Thr22, Ser24, Asp36, Ser87, Ile107, Lys170, Met199, Ser204, or Lys213 of the amino acid sequence of naturally produced Bacillus amyloliguefaciens subtilisin, wherein said modified subtilisin has altered thermal stability in comparison to said precursor subtilisin.
 14. The modified subtilisin of claim 13 wherein said precursor subtilisin is a recombinant subtilisin.
 15. The modified subtilisin of claim 13 comprising a substitution of at least two of said amino acid residues of said precursor subtilisin.
 16. The modified subtilisin of claim 13 wherein said modified subtilisin is substantially isolated.
 17. A modified subtilisin comprising a substitution of at least one amino acid residue in the amino acid sequence of a precursor subtilisin in at least one position equivalent to Ser24, Asp36, Met50, Ile107, Met124, Glu156, Gly166, Gly169, Lys170, Asp197, Ser204, Lys213, Tyr217, or Met222 of the amino acid sequence of naturally produced Bacillus amyloliguefaciens subtilisin, wherein said modified subtilisin has altered alkaline stability in comparison to said precursor subtilisin.
 18. The modified subtilisin of claim 17 wherein said precursor subtilisin is a recombinant subtilisin.
 19. The modified subtilisin of claim 17 comprising a substitution of at least two of said amino acid residues of said precursor subtilisin.
 20. The modified subtilisin of claim 17 wherein said modified subtilisin is substantially isolated.
 21. A modified subtilisin comprising a substitution of at least one amino acid residue in the amino acid sequence of a precursor subtilisin in at least one position equivalent to His67, Ile107, Glu156, Gly166, Lys170, Lys213, or Met222 of the amino acid sequence of naturally produced Bacillus amyloliguefaciens subtilisin, wherein said modified subtilisin has an altered pH activity profile in comparison to said precursor subtilisin.
 22. The modified subtilisin of claim 21 wherein said precursor subtilisin is a recombinant subtilisin.
 23. The modified subtilisin of claim 21 comprising a substitution of at least two of said amino acid residues of said precursor subtilisin.
 24. The modified subtilisin of claim 21 wherein said modified subtilisin is substantially isolated.
 25. A composition comprising the modified subtilisin of any one of claims 1-4 in combination with a detergent.
 26. A composition comprising the modified subtilisin of any one of claims 5 or 6-8 in combination with a detergent.
 27. A composition comprising the modified subtilisin of any one of claims 9 or 10-12 in combination with a detergent.
 28. A composition comprising the modified subtilisin of any one of claims 13 or 14-16 in combination with a detergent.
 29. A composition comprising the modified subtilisin of any one of claims 17 or 18-20 in combination with a detergent.
 30. A composition comprising the modified subtilisin of any one of claims 21 or 22-24 in combination with a detergent.
 31. A nucleic acid encoding the modified subtilisin of claim 1 or
 2. 32. A nucleic acid encoding the modified subtilisin of any one of claims 5, 6 or
 7. 33. A nucleic acid encoding the modified subtilisin of any one of claims 9, 10, or
 11. 34. A nucleic acid encoding the modified subtilisin of any one of claims 13, 14, or
 15. 35. A nucleic acid encoding the modified subtilisin of any one of claims 17, 18 or
 19. 36. A nucleic acid encoding the modified subtilisin of any one of claims 21, 22 or
 23. 37. An expression vector comprising the nucleic acid of claim
 31. 38. An expression vector comprising the nucleic acid of claim
 32. 39. An expression vector comprising the nucleic acid of claim
 33. 40. An expression vector comprising the nucleic acid of claim
 34. 41. An expression vector comprising the nucleic acid of claim
 35. 42. An expression vector comprising the nucleic acid of claim
 36. 43. A host cell transformed with the expression vector of claim
 37. 44. A host cell transformed with the expression vector of claim
 38. 45. A host cell transformed with the expression vector of claim
 39. 46. A host cell transformed with the expression vector of claim
 40. 47. A host cell transformed with the expression vector of claim
 41. 48. A host cell transformed with the expression vector of claim
 42. 